Fiber sampler for recovery of bioaerosols and particles

ABSTRACT

An aerosol collection system and method. The system includes a bio-aerosol delivery device configured to supply bioparticles in a gas stream, a moisture exchange device including a partition member coupled to the gas stream and configured to humidify or dehumidify the bioparticles in the gas stream, and an aerosol collection medium downstream from the moisture exchange device and configured to collect the bioparticles. The method includes delivering bioparticles in a gas stream, humidifying or dehumidifying the bioparticles in the gas stream by transport of water across a partition member and into a vapor phase of the gas stream, and collecting the bioparticles by a collection medium.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority under 35 U.S.C. §119(e) to U.S. Ser. No. 61/600,366 filed Feb. 17, 2012 entitled“Improved Fiber Sampler for Recovery of Bioaerosols and Particles,” theentire contents of which are incorporated herein by reference. Thisapplication is related to PCT/US2011/048094, filed Aug. 17, 2011,entitled “Fiber Sampler for Recovery of Bioaerosols and Particles,” theentire contents of which are incorporated herein by reference. Thisapplication is related to U.S. Application Ser. No. 61/374,466, filedAug. 17, 2010, entitled “Fiber Sampler for Recovery of Bioaerosols andParticles,” the entire contents of which are incorporated herein byreference. This application is related to U.S. application Ser. No.13/211,940, filed Aug. 17, 2011, entitled “Fiber Sampler for Recovery ofBioaerosols and Particles,” the entire contents of which areincorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under HSHQDC-09-C-00154awarded by DHS. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention is related to fibers, methods, and devices for collectionof bioaerosols and particles on fiber structures. The invention is alsorelated to electrospun materials for filtration and air sampling, inparticular the collection of bioaerosols.

Description of the Related Art

This application is related to U.S. application Ser. No. 11/559,282,filed on Nov. 13, 2006, entitled “Particle Filter System IncorporatingNanofibers,” the entire contents of which are incorporated herein byreference. This application is related to U.S. application Ser. No.10/819,916, filed on Apr. 8, 2004, entitled “Electrospinning of PolymerNanofibers Using a Rotating Spray Head,” the entire contents of whichare incorporated herein by reference. This application is also relatedto U.S. application Ser. No. 10/819,942, filed on Apr. 8, 2004, entitled“Electrospray/electrospinning Apparatus and Method,” the entire contentsof which are incorporated herein by reference. This application isrelated to U.S. application Ser. No. 10/819,945, filed Apr. 8, 2004,entitled “Electrospinning in a Controlled Gaseous Environment,” theentire contents of which are incorporated herein by reference. Thisapplication is related to U.S. Ser. No. 11/130,269, filed May 17, 2005entitled “Nanofiber Mats and Production Methods Thereof,” the entirecontents of which are incorporated herein by reference.

Collection of both indoor and outdoor air samples is important formonitoring air quality. A wide range of microorganisms are of interestincluding bacteria, fungi and viruses. From a health standpoint, toxinsand allergens may be of interest as well. For example see, J. M. Macher(1999) Bioaerosols, Assessment and Control, American conference ofGovernmental Industrial Hygienists, Cincinnati, Ohio.

More recently, concerns about airborne pathogens being present due tonatural processes, accidents, or terrorist attacks has led to the needfor improved sampling systems. In addition to the problem of collectingthe aerosol (particles) is the problem of recovering the particles foranalysis. In the case of biological particles, a common problem is thatthe organisms die during collection or after collection while awaitinglaboratory analysis. Current sampling methods onto microbiological mediado not permit extended sampling times beyond 30-45 minutes in the casewhere preservation of viable organisms is of interest.

In general, a concentrated, viable collect of submicrometer biologicalparticles has been recognized in the art as a challenge. Each bioaerosolsampling method has limitations with respect to sampling time,desiccation, shelf life of sample, complexity, compatibility withanalysis via PCR and live recovery. Some evaluations are given byGriffiths and Decosemo (1994); Henningson and Ahlberg (1994); Wang,Reponen et al. (2001); Tseng and Li (2005); Verreault, Moineau et al.(2008); Mainelis and Tabayoyong (2010) listed below:

-   Griffiths, W. D. and G. A. L. Decosemo 1994. The Assessment of    Bioaerosols—a Critical-Review. Journal of Aerosol Science 25(8):    1425-1458.-   Henningson, E. W. and M. S. Ahlberg 1994. Evaluation of    Microbiological Aerosol Samplers—a Review. Journal of Aerosol    Science 25(8): 1459-1492.-   Mainelis, G. and M. Tabayoyong 2010. The Effect of Sampling Time on    the Overall Performance of Portable Microbial Impactors. Aerosol    Science and Technology 44(1): 75-82.-   Tseng, C. C. and C. S. Li 2005. Collection efficiencies of aerosol    samplers for virus-containing aerosols. Journal of Aerosol Science    36(5-6): 593-607.-   Verreault, D., S. Moineau and C. Duchaine 2008. Methods for sampling    of airborne viruses. Microbiology and Molecular Biology Reviews    72(3): 413-444.-   Wang, Z., T. Reponen, S. A. Grinshpun, R. L. Gorny and K.    Willeke 2001. Effect of sampling time and air humidity on the    bioefficiency of filter samplers for bioaerosol collection. Journal    of Aerosol Science 32(5): 661-674.

The collection of bioaerosols is currently performed by a number ofdevices that have been available for quite some time. Common bioaerosolsampling devices include:

-   -   Impactors where a jet of air deposits the bioaerosol particle on        a media surface.    -   Impingers where the jet of air impinges on a surface within a        liquid filled container.    -   Filters where the particles are collected on the surface of the        filter.

Impactors are limited with respect to sampling time because thecollection media used to enumerate the number of colonies of organismsfor viability after collection is subject to desiccation, thus limitingthe sampling time. Also typical impactors designed for microorganismshave a lower particle size collection limit of about 0.5 micrometers.(Anderson, A. A. (1958) New sampler for collection, sizing, andenumeration of viable airborne particles, J. Bacteriol. 76, 471-484)

Impingers are limited in their sampling time from the evaporation of thecollecting fluid. The collection efficiency is dependent on the volumeof fluid in the impinger. Also the microorganisms may be lost byreaerosolization from the fluid during sampling (Grinshpun, S. A., K.Willeke, V. Ulevicius, A. Juozaitis, S. Terzieva, J. Gonnelly, G. N.Stelma and K. P. Brenner (1997) Effect of impaction, bounce andreaerosolization on the collection efficiency of impingers. Aerosol Sci.Technol. 26, 326-342).

Filters and other collection media such as membranes have long been usedto trap aerosol and bioaerosols for subsequent analysis thereof. Filterswith a poor figure of merit or quality at least require higher pressuresto force air flow through. An example consequence is that in portablesamplers operation is severely limited due to battery life in thesamplers with filters with high pressure drop. Filter figure of merit orquality is defined as FoM=−log(Pt)/ΔP, where Pt is the penetration ofparticle at a specific size through the filter and ΔP is the pressuredrop at a specific gas flow rate. The larger the FoM, the better will bethe performance of the filter. See Hinds, W. C. (1982) AerosolTechnology, Wiley, New York, N.Y.). Further, the flow of air through thefilters or membranes after a biological aerosol has been trapped canlead to the desiccation of the medium about the bioaerosol and death ofthe bioaerosol.

Thus, in general, a list of existing air sampling technologies forbioparticles and their drawbacks are provided below.

Typical longest Sampler d₅₀ sampling time Notes Impinger ~0.3 μm 30 minGood for short e.g. AGI-30 term sampling Impactor ~0.7 μm 20 minCollection on e.g. Anderson agar reduces desiccation SKC ~0.3 μm 8 hrsFluid for long BioSampler term sampling interferes with PCR Filtration *60 min^(†) Desiccation is a e.g. 37-mm significant cassette with problemwith Nucleopore filtration * Filtration has a most penetrating sizeabout 0.1 to 0.3 μm with efficiency of collection typically high (>80%)across size range. ^(†)Longer term sampling is possible but organisms donot survive.

SUMMARY OF THE INVENTION

In one embodiment of the invention, there is provided an aerosolcollection system including a bio-aerosol delivery device configured tosupply bioparticles in a gas stream, a moisture exchange deviceincluding a partition member coupled to the gas stream and configured tohumidify or dehumidify the bioparticles in the gas stream, and anaerosol collection medium downstream from the moisture exchange deviceand configured to collect the bioparticles.

In one embodiment of the invention, there is provided a method forcollecting aerosols. The method includes delivering bioparticles in agas stream, humidifying or dehumidifying the bioparticles in the gasstream by transport of water across a partition member and into a vaporphase of the gas stream, and collecting the bioparticles by a collectionmedium.

It is to be understood that both the foregoing general description ofthe invention and the following detailed description are exemplary, butare not restrictive of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings, wherein:

FIG. 1 is a table showing sampling challenges for the sampling andpreservation of bioaerosols;

FIGS. 2A and 2B are schematics of fiber structures of the invention;

FIG. 3 is a schematic showing the collection of bioaerosol particles ina nanofiber filter;

FIG. 4 is a schematic showing a combination of a humidifying sectionfollowed by a fiber filter of the invention;

FIG. 5 is a schematic showing the fiber filter with injection of waterinto the fiber filter to maintain an environment on the filter of 70% RHin this one illustrative example;

FIG. 6 is a schematic of applying fiber collection surfaces tobioaerosol collection with an impactor;

FIG. 7 is a schematic of the combination of humidification of thebioaerosol with an impactor containing fibers on the collection surface;

FIG. 8 is a schematic showing a cascade impactor with fiber collectionsurfaces and water introduction to maintain a controlled relativehumidity;

FIG. 9 is Table 1 depicting an example of polymer and surfacechemistries studied in this invention;

FIG. 10 is a SEM micrograph of a perspective view of a nanofiberstructure formed by deposition of PU on a part of a web material;

FIG. 11A is another SEM micrograph of a perspective view of a nanofiberstructure formed by deposition of PU on web material showing nanofibercoverage and orientation over an opening in the underlying web material;

FIG. 11B is another SEM micrograph showing a cross section of ananofiber structure formed by deposition of PU on web material;

FIG. 11C is another SEM micrograph of a nanofiber structure formed bydeposition of PU on web material;

FIG. 11D is a composite view showing differently scaled depictions of ananofiber sampling filter in a 37 mm cassette format;

FIG. 12A is a composite of two scanning electron micrograph SEM imagesshowing a collection including a Bacillus globigii (Bg) spore and whatare likely MS2 virus particles;

FIG. 12B is a graph of pressure drop curves (pressure drop versus facevelocity) for two common commercial air sampling filter materials ascompared with nanofiber filter media composed of PSU or PU depositedthereon;

FIG. 13 is Table 2 depicting a comparison of the fiber filter mats ofthe invention to a standard Teflon filter using the virus MS2;

FIG. 14 is a comparison of viabilities between fiber filter mats of theinvention and gelatin and Teflon filters where a bioaerosol of Serratiawas sampled for 3 hours;

FIG. 15 is a comparison of viabilities between fiber filter mats of theinvention and gelatin where a bioaerosol of Serratia was sampled, andshows the impact of sampling face velocity on the viability of Serratia;

FIG. 16 is a depiction showing of the viabilities obtained whencollecting fragile Yersinia rohdei using RH controlled filtration withthe fiber filter mats of the invention;

FIGS. 17A and 17B are Tables 3 and 4 depicting organism survivability ondifferent surfaces and different environmental conditions;

FIG. 18 is a depiction showing the storage of the slightly fragileStaphylococcus and very fragile organism Yersinia at different storageconditions;

FIG. 19 is a schematic depiction of a sample storage deviceincorporating a moisture providing material;

FIG. 20 is a schematic depiction of another sample storage deviceincorporating a moisture providing mechanism;

FIG. 21 is a scatter plot of metrological year data showing the range oftemperature and relative humidity condition;

FIG. 22 is a psychrometric chart showing potential ways of conditioningthe bioaerosol sample to the ideal sampling and preservation conditions;

FIG. 23 is a psychrometric chart illustrating preferred paths forconditioning the bioaerosol sample to avoid killing the organisms;

FIG. 24A is a schematic diagram depicting a bioaerosol sampling systemof this invention;

FIG. 24B is a schematic diagram depicting a bioaerosol sampling systemof this invention illustrating the principle of multiple or cascadedmoisture devices with the final device in the temperature controlledvolume at sampler/filter conditions;

FIG. 25A is a depiction of a humidity control system of this invention;

FIG. 25B is a depiction of another humidity control system of thisinvention;

FIG. 25C is schematic diagram depicting a bioaerosol sampling system ofthis invention similar to the system shown in FIG. 24B with multiple orcascaded moisture devices;

FIG. 25D is a schematic depicting humidity conditioning for a singleRH-conditioned biosampler;

FIG. 25E is a schematic depicting two-stage humidity and temperatureconditioning for a biosampler;

FIG. 26 is an illustration of the tubular membrane moisture exchanger asused to create controlled relative humidity conditions extending theconcept in FIG. 6;

FIG. 27 is an illustration of the applied of controlled temperature andcontrolled relative humidity for sampling bioaerosols for preservationextending the concept in FIG. 26:

FIG. 28 is a depiction of the tubular membrane moisture exchanger whencold dry air is used to condition warm moist bioaerosol;

FIG. 29 is a depiction of the tubular membrane moisture exchanger whencold moist air is used to cool and humidify hot dry bioaerosol;

FIG. 30 is a depiction of the tubular membrane moisture exchanger whenwarm moist air is used to heat and humidify cold dry bioaerosol; and

FIG. 31 is a depiction of cascade of heater/coolers and tubular membranemoisture exchangers to control the temperature and relative humidity ofthe bioaerosol to compensate for water condensation or undue dryness;

FIG. 32 is a schematic illustration of a computer system forimplementing various embodiments of this invention including control ofthe bioaresol collection; and

FIG. 33 is an illustration of another cascade of conditioning steps tocontrol the temperature and humidity of the bioaerosol to compensate forwater condensation and undue dryness, showing pathways for the aerosolwith minimum expansion and contraction of the flow to preventbioparticle losses.

DETAILED DESCRIPTION OF THE INVENTION

As described below, this invention addresses collection medium for thecapture and storage (under viability enhancing conditions) bioparticlesand also addresses three aspects of bioparticle collection and storagewhich also improve the viability. The particular aspects of bioparticlecollection and storage addressed include (but are not limited to):

(1) collection aided by impaction onto nanofiber substrates undercontrolled relative humidity conditions, as described for example inU.S. application Ser. No. 13/211,940 noted above;

(2) direct filtration (flow-through) with relative humidity RH controlbut at ambient temperatures; and

(3) the simultaneous RH and temperature conditioning during sampling.

As used herein, “bioparticles” means microbes and other biologicalparticles such as for example bacteria, viruses, and biologicallyderived particles such as proteins, cell fragments, etc.

As used herein, “viable” or “viability” is defined as the capability ofhaving a collected organism becoming active again after being placedinto a favorable environment. For example, a collected bacteria spore orvegetative bacterium being placed into a growth media and incubatedunder appropriate conditions for growth resulting in growth andreproduction of the organism. For example, a collected virus beingexposed to its desired host and incubated under appropriate conditionsresulting in the virus infecting the host.

As used herein, “collection viability” means the capability to keep apercentage of bioparticles in a collection medium of this inventionalive during the collection event.

As used herein, “storage viability” means the capability to keep apercentage of bioparticles in a collection medium of this inventionalive from the time of collection until the bioparticles are analyzed orcounted.

As used herein, “viability enhancement” or “enhanced viability”encompasses both collection viability and storage viability and meansthe capability to collect a percentage of bioparticles from a mediumwithout death and keep the collected bioparticles alive until thebioparticles are analyzed or counted.

As used herein, an “osmotic material” is as any material that has thecapacity to provide transport of liquids (such as for example water ornutrients) to or from the collected bioparticles. For example, a fibercomposed of a hydrophilic polymer would represent on one kind of osmoticmaterial.

As used herein, “design limiting” organisms are organisms which areextremely fragile and extremely difficult to keep alive.

Viable Sampling

In order to determine if an organism is infectious for the purpose ofmaking health related decisions, the viability must be assessed byculture methods where the presence of live organisms at the start of theculture is needed.

Maintaining viability during and after collection is well known to be achallenge. Some organisms are very hardy, such as bacterial spores.These organisms can be very difficult to kill. The same traits that makethem difficult to kill make them more readily kept alive or viableduring collection and storage. Other organisms are extremely fragile andextremely difficult to keep alive. Maintaining viability of these designlimiting microorganisms during collection and during storage is achallenge. Organisms lose viability during collection due to desiccationby either the air moving past these organisms during the collectionprocess or from a process such as evaporation. Also, any condition thatleads to an increase in hydroxyl radicals will decrease viability.

Thus, collection of bacteria and virus (microbes) while keeping thesebioparticles viable in the case of long term sampling is problematic.

Referring now to the drawings, wherein like reference numerals designateidentical or corresponding parts throughout the several views, FIG. 1details the challenges overcome by the invention for representativeorganisms important from a health standpoint. More specific, challengesdetermined by the inventors for conducting long term air sampling, butare not restricted to long term air sampling, in the collection ofviable bioparticles include:

-   -   Viability during sampling    -   Sampling duration time    -   Viability of collected sample during storage    -   Compatibility of collected sample on the collection medium with        analysis techniques.        Bioparticle Sample Collection Devices

Electrospun micro and nanofibers from polymer solutions provide a highsurface area environment with tunable surface chemistries which can beconducive to the collection and retention of biological particles.Indeed, the invention in one embodiment provides a sampling device forthe collection and recovery of particles, including biological particlessuch as bacteria, viruses, and yeasts. The sampling device provides forenhanced viability of biological particles and provides for quantitativerecovery of samples for laboratory analysis, as detailed below.

The invention provides for a device, based on a fiber mat or a nanofibermat that provides for collection of bioparticles including bacteria,fungi, viruses, and other biological particles (e.g., bioaerosols). Thecollection is achieved in one aspect of the invention either through theuse of the fiber mat as a filter, for example a high efficiency lowpressure drop nanofiber flow-through filter, through the use of thefiber mat as a substrate for impaction of particles, or for the use of afiber mat as a wipe.

In one embodiment of the invention, the bioparticles are kept viable forextended periods of time (e.g., 1 day to >7 days) without extraordinaryefforts because the biological particles are collected in amoisture-rich (or nutrient-rich) fiber mat or nanofiber filter mat.Furthermore, samples can be recovered from the mats for analysis byextraction in buffer or other suitable liquid. Alternately, the fiberscan be configured to be dissolved using, for example a low acid orenzymatic solution. Indeed, in one embodiment of the invention, the nanoor microfiber material can be constructed from polymers that provide fordissolution in water or an appropriate buffer. Such capability canimprove recovery of collected bioparticles for culture and non-cultureanalysis method such as PCR (polymerase chain reaction), ELISA(Enzyme-Linked Immunosorbent Assay), and a variety of other molecularand biochemical techniques.

In one embodiment of the invention, the fibers are deposited on avariety of backing materials which could include moisture absorbingproperties or ability to provide moisture to the fiber mat; for example,super-soaker polymers, hydrophilic polyurethane foam, blotter paper,polymer nonwoven mats containing hydroscopic salts such as lithiumchloride, and related methods. Accordingly, the fibers of the inventioncan form in one embodiment a bioparticle collection device including acollection medium including a plurality of fibers formed into a fibermat and an osmotic material disposed in contact with the plurality offibers.

In one embodiment of the invention, the structure and surface chemistryof the fibers, incorporation of additives, or mixed fiber materialsincorporating osmotic materials can contribute to the collection andpreservation of the bioparticles. Furthermore the container or packagingof the fiber material can aid in preservation. For example, a sealedcontainer containing a hydrogel or other material can be used tomaintain RH to aid in viability preservation.

U.S. Pat. No. 4,805,343 (the entire contents of which are incorporatedherein by reference) describes for example cellulose acetate hollowfibers that have osmotic properties. Such fibers (or other hydrophilicfibers) could be used in the present invention to provide an externalsupply of water or nutrients transported to the fiber mats collectingthe bioaerosols. Alternatively, cellulose acetate fibers could beintermixed into the fiber mats collecting the bio-aerosols.

The use of a fibrous matrix to collect and preserve the bioparticlesalso provides advantages from the equipment design and operation pointof view. A long term (>8 hrs) liquid-based sampler typically wouldrequire a fluidics system to remove sample and replenish buffers. AnRH-controlled fibrous material format would not require as an extensivefluidics system. Furthermore, if a large amount of dust, pollen, andother small particles are present in the fluidics system, then theinstrument could become clogged.

A fibrous matrix approach that is free of a fluidics system couldtolerate samples laden with dust, but these particles would not shut thesystem down. Additionally, the mass of liquid needed to operate a systemlong term could be significantly less. The weight and complexity of alow liquid use or nearly liquid-free sample collection/preservationsystem could also be much less compared to a liquid collection system.

In one embodiment of the invention, the fibers are deposited on variousbacking materials, and the combination of the fibers and the backingmaterials is used as an impaction substrate for collection of theaerosol. For example, the fibers can be electrospun onto a foil andplaced as a part of the impaction plate in a standard impactor for airsampling.

The fiber matrix (and especially a nanofiber matrix) provides a highsurface area environment for collecting organisms. At the micro-scale ofbacteria and viruses, surface chemistry can be important. Using polymersprovides for adjustable surface chemistries from hydrophilic tohydrophobic. Furthermore, hydrogels including polymer networks thatreadily hold water can be used to regulate the moisture content of thenanofiber matrix. Examples of such systems include polymers of acrylicacid combined with sodium hydroxide and co-polymers of poly(2-hydroxyethyl methacrylate) (polyHEMA). Complex multi-fiber and layeredstructures can easily be fabricated to provide a mixed environment thatcannot be obtained with a liquid system. This mixed environment canpotentially provide a way for a variety of organisms that preferdifferent environmental conditions to exist in the same sample. Anexample of a mixed environment is simultaneously electrospinning twodifferent polymers onto a common collection substrate thus creating afibrous mat with two different polymers which would have two differentsurface chemistries and/or fiber diameters.

In one embodiment of the invention, the fibrous matrix sample collectiondevice includes mechanisms such as those described above or othermechanisms to provide moisture or to maintain the RH in a desired range,for example from 65% to 85% or more precisely 70% to 85% or moreprecisely 75% to 81%.

In one embodiment of the invention, a polyurethane PU fiber, thestructure of the PU nanofibers, the corresponding nonwoven, and the RHall contribute to viability maintenance. In this embodiment, theviability enhancing aspect appears to be only the PU nanofiber mat andsurface humidity of the nanofibers, and there is no need for anadditional osmotic material, although such an addition could be used.

In one embodiment of the invention, the sample collection deviceprovides viable storage at ambient temperature and RH. In anotherembodiment of the invention, viability maintenance is enhanced,especially for particularly fragile organisms, via storage at cooledconditions. Storage of fragile organisms such as Yersinia has beendemonstrated for more than 9 days when stored on a polyurethane (PU)nanofiber media in a laboratory refrigerator.

On one hand, while keeping collected organisms wet may result ingermination or growth, and the collection conditions might be good forone class or organisms, the collection conditions might be bad foranother class of organisms. On the other hand, an overly dry environmentcan also kill organisms. A fibrous matrix (optionally combined withother humidity control devices) can provide a relative humidity(RH)-controlled environment to improve preservation of viability ofbioaerosols while potentially simplifying sample handling and storage.

Filter Collection Systems

Nanofibers can be used in one embodiment of the invention as a lowpressure drop, high efficiency collection filter in any standardsampling form such as the commercial ‘37 mm air monitor cassette’ orother sampling cassette device. (The nanofibers can also be used in animpaction device for example in an eight-stage impactor.)

FIGS. 2A and 2B show schematics of various fiber structures. Thedifferent fibers designations indicate different function inmicroorganism preservation. In one embodiment, fibers are present thatcontain or regulate water moisture. For example, the fibers such ashydrogel polymers or crosslinked polyHEMA or gelatin or similar suchmaterial can be used as for hydration. Alternate versions includehydrophilic polymers, like cellulose and its derivatives (e.g. celluloseacetate) and may include incorporation of hydroscopic salts such aslithium chloride; or hydrogel particles, such as those formed fromacrylic acid combined with sodium hydroxide, with these particlesentrapped in the fiber matrix. In FIGS. 2A and 2B, the white spacebetween the two designated types of fibers represents air space or otherfibers in the collection mat (for example having a density and size topromote collection viability and storage viability of a bioaerosol).

In one embodiment, the white space between the two designated types offibers may be filled with particles which themselves contribute to theviability of the collected bioaerosols. These particles can beintroduced during the electrospinning process in a manner as describedin U.S. Pat. No. 7,297,305 (the entire contents of which areincorporated herein by reference). For example, particles (e.g.,antioxidant particles or nutrient particles) which can contribute to theviability of the collected bioaerosols can be introduced into the fluidssuitable for electrospraying and/or electrospinning. Alternatively,these particles can be introduced in a manner as described in U.S. Pat.Appl. No. 2006/0264140 (the entire contents of which are incorporatedherein by reference).

In this process, particles which can contribute to the viability of thecollected bioaerosols are delivered into a fiber-extraction region of anelectrospinning apparatus. The introduced particles collide and combinewith the electrospun fiber material during formation of the fibers andthe fiber mat. Alternatively, these particles can be introduced afterthe electrospinning process by flowing a solution (non-reactive with thefibers in the fiber mat and containing the particles of interest)through the fiber mat. The solution can be thereafter evaporated orretained if the solution itself is a substance which can contribute tothe viability of the collected bioaerosols.

The fibers in FIGS. 2A and 2B may be aligned or may have randomorientations. The fibers in FIGS. 2A and 2B would in one embodiment bein contact with one another in the fiber mat.

In one embodiment of the invention, the hydration fibers are not berequired. In one embodiment of the invention, the preservation fibersare not required. When used, the preservation fibers, due to theirsurface chemistry and structure, promote preservation of thebioparticles. A more detailed description of preserving fibers isprovided below.

Accordingly, FIG. 2A illustrates intermixed fiber material made bysimultaneous electrospinning onto a common collection plane, and FIG. 2Billustrates the concept of a layered structure that can be formed eitherby sequential electrospinning to make a layered structure or by spinningfrom opposing directions to a common plane to simultaneously build totwo sides of the composite, layered structure.

As noted above, the fiber mat of the invention can be configured as animpaction substrate or as a flow though filter, and can be used in avariety of air sampling systems and configurations.

Methods of Conditioning Bioparticle Prior to Collection

In one aspect of the invention, the conditioning of inlet air containingbioparticles facilitates the collection of viable bioparticles. In oneaspect of the invention, the collection of the bioparticles occurs ontoan appropriate substrate (media) that aids in collection of viablebioparticles, aids in storage of the viable bioparticles, and permitsanalysis via a variety of techniques (e.g. live culture, PCR-basedanalysis methods, immuno-based assays, etc.).

Accordingly, in one embodiment of the invention, a bioparticle isexposed to the vapor or a working fluid (for example biocompatiblefluids such as water). Subsequently, vapor condensation ontobioparticles is induced by either adiabatic expansion or cooling, or bymixing with a cooler airflow.

Accordingly, in one embodiment of the invention, the formedparticle-water-condensate bioparticles are collected on the collectionmedium of the invention.

FIG. 3 is a schematic showing the collection of bioaerosol particles ina fiber filter. The collection in the fiber filter occurs byinterception, impaction, and diffusion. In one embodiment, a nanofiberfilter has low pressure drop and high efficiency and creates anenvironment for preservation of microorganisms.

FIG. 4 shows a fiber filter following a humidifying section whichcontrols the humidity at the fiber filter at a target value or range,for example 50 to 85% RH. The humidification chamber (in one embodiment)is disposed at the site of the mixing of humidified air with thebioaerosol sample. The humidification chamber (in another embodiment) isa chamber where water is introduced into the air by wetted porous wallsto maintain e.g., a relative humidity of 70 to 80% at the filter.

FIG. 5 shows the arrangement of introducing water into the fiber filterto maintain an environment e.g. a relative humidity of 70 to 80% whichpreserves microorganisms during sampling. The humidity wouldalternatively be maintained during storage.

FIG. 6 shows combination of the fiber with a cascade impactor. Thefibers, including nanofibers, prevent rebound of the particles and canprovide an environment to preserve the microorganisms. The impactorshave small holes forming jets of air directing particles at thecollection stage at a high velocity (usually less than 0.3 Mach). Theinertia of the particles causes the particles to impact on the fibers.

FIG. 7 shows an impactor containing fiber collection on collectionstages with a humidification of the bioaerosol. Humidification (in oneembodiment) involves the mixing of the air containing bioaerosol withmoist air or (in another embodiment) evaporation of water within thehumidification chamber from wet porous walls.

FIG. 8 shows the introduction of water into an impactor with fiber onthe collection surface to maintain a controlled humidity e.g., at 70%RH.

Accordingly, in this invention, there are provided a number of ways forconditioning of bioparticles prior to collection, adding water moistureto the sampled air stream, and regulating the relative humidity (RH) ofthe sampled air stream. Addition of water moisture or regulation of theRH can be achieved via a number of methods including use of a wet walledtube to provide humidity to the sampled air, atomization of water toprovide humidity to the sampled air, mixing a wet or dry air stream withsampled air stream to provide air stream at target RH (wet air could begenerated through bubbling air through water, a wet walled tube,atomization of water, etc.), and other ways to regulate the RH of asampled air stream.

Methods of Making Fiber Substrates for Bioparticle Collection

Electrostatic spinning of polymer solutions to form micro and nanodiameter fibers, better known as electrospinning, is a ready method tomake nonwoven fibrous mats. In one embodiment of this invention,electrospinning is used to make fibrous mats but other methods offabricating mats of micro and nanofibers may also be a route to formfibrous structures described in this invention. U.S. Pat. Nos. 5,494,616and 6,520,425; and Badrossamy M R et al., Nano Letters 2010, 10(6):2257both describe alternative techniques applicable to the invention. Theentire contents of these documents are incorporated herein by reference.

A wide variety of polymers can be electrospun into fibers including bothsynthetic polymers such as polystyrene and natural polymers such ascollagen and gelatin. Polymers offer hydrophobic to hydrophilic surfaceproperties including functionalities similar to sugars or proteins. FIG.9 shows in Table 1 only a limited number of polymer and surfacechemistries that are suitable for this invention.

In terms of the use of electrospun fibers for filter mats suitable forthe invention, U.S. Pat. Appl. Publ. No. (2005/0224999), the entirecontents of which are incorporated herein by reference, describes theuse of an electronegative gas to facilitate the electrospinning processby the introduction, for example, of carbon dioxide (CO₂) around thespinning orifice or emitter. Gases such as CO, CF₄, N₂O, CCl₄, CCl₃F,CCl₂F₂ and other halogenated gases can be introduced into theelectrospinning environment. These electronegative gases stabilize theTaylor cone formed by the polymer jet as it comes off the needle,reduces corona discharge at the needle, and reduces fiber diameter.Furthermore, spinning in a controlled environment ensures lesscontamination of the fibers, improves safety, and adds another dimensionof control parameters that can be used to fine-tune fiber formation.

An electronegative gas can be passed coaxially with the spinning needlealong with use of a controlled gas environment. Typically, a gas shroudis used to provide the coaxial gas flow. A typical shroud can be in theshape of an annulus having an outside radius of about 0.48 cm and aninside radius of about 0.40 cm. Insulating and metallic shroud memberscan be used. A variety of geometries and sizes are possible; such as forexample a circular outside with a hexagonal inside being an additionalgeometry. In the annular geometry, a distance from an exit end of theannulus where gas is emitted to the tip of the electrospinning elementcan range from flush (0 cm) to 8 cm; with a typical distance beingaround 4 to 5 cm, and with the distance being 4.7 cm for the detailedexamples later.

Control of the electrospinning conditions has produced polymernanofibers with an average fiber diameter AFD of 100 nm and less.Nanofibers less than 400 nm have been found to improve the filtrationproperties of the resultant fiber when combined with other elements ofthe invention.

Additives in the polymer solution can make a substantial difference infiber size and quality. Addition of trace amounts of a salt or asurfactant increases the solution conductivity and hence the chargeaccumulation at the tip of the electrospinning element resulting inlarger stretching forces applied to the forming fiber, hence smallerdiameter fibers. The surfactant also reduces the surface tension of thepolymer allowing for even smaller fibers to be spun. Lithium salts, (forexample, lithium chloride and lithium triflate) or surfactants such astetra butyl ammonium chloride (TBAC) are suitable for the invention.Lithium salt concentrations from 0.01 to 3 wt % are suitable for theinvention. Concentrations of TBAC of between 0.06 and 0.4 wt %, wereexemplary, although other concentrations are suitable.

Stainless steel extrusion tips from 0.15 mm to 0.59 mm internaldiameters (ID) are suitable for the invention. Larger and smallerdiameters may also be used. Teflon™ capillary tubes with ID from 0.076mm to 0.31 mm are suitable for the invention. Larger and smallerdiameters may also be used. Both types of orifices can produce smallfibers. For both orifices, low flow rates of the polymer solution (e.g.,0.05 ml/hr) coupled with high voltage drops typically resulted in thesmallest fiber diameters (e.g., AFD less than 100 nm). In both cases,the voltage was set to 22 kV to 30 kV for a 17.8 cm to 25.4 cm gap(i.e., distance between emitter 2 and mesh 7). Of note is that thevoltage per electrospinning-gap is one parameter determining the pullingstrength; this gap also determines a travel time thus partly determiningfiber stretching time.

Besides stainless steel and Teflon™ extrusion tips, in the invention,other materials (provided the materials are non-reactive with thesubstance being electrospun including any solvent used in theelectrospinning process) can be used such as for example polymers,glass, ceramic, or metal extrusion tips.

The relative humidity RH of the electrospinning chamber effects fibermorphology. For example, when using 21 wt % PSu (M_(w)˜35,000 g/mol) inDMAc, a high RH (e.g., >65%) resulted in fibers that have very fewdefects and smooth surfaces but larger diameters. A defect in a fiber isin general seen as a deviation from a smooth round fiber of long length.Defects thus are beads on the fiber, variations in fiber diameter in theaxial direction, etc. A low RH (e.g., <13%) resulted in smaller fibersbut more defects. Modestly low RH (e.g., 40% to 20%) typically producedsmall fiber size with fewer defects.

A variety of mechanisms are suitable in the invention to control thechamber RH such as placing materials that absorb (e.g. calcium sulfate)or emit water moisture (e.g., hydrogels), operating a small humidifierin the chamber, and adding moisture into the process gas streams priorto introduction to the electrospinning chamber. For example, positiveresults were obtained by bubbling CO₂ through deionized (DI) water andthen introducing the humidified CO₂ gas into the chamber. In oneembodiment of the invention, two gas streams (e.g., one humidified andone dry) are used to obtain a desired RH for the chamber and/or for thegas jacket flowing over the electrospinning orifice.

The fiber diameter obtained in the invention is a function of thepolymer molecular weight, the polymer architecture, the solvent orsolvents, the concentration of polymer in the solvent system, theadditives and their concentration, the applied electrospinningpotential, the gap between the spinning orifice and ground, the size andshape of the spinning orifice, the polymer solution flow rate, the flowrate and composition of the process gas that flows over the needle, theRH of the process gas, and the partial pressure of the solvent(s).

Other embodiments of the invention could use different polymer solventsystems and hence different electrospinning conditions to obtainappropriate nanofibers. Furthermore, the same polymer solvent systemscould be combined with different electrospinning conditions to createimproved fibers or fibers tailored for alternative applications. Forexample, the jacket of CO₂ gas flowing over the needle could containsolvent vapor in order to lower the evaporation rate of the solvent(s)in the polymer jet formed at the needle tip, thus increasing stretchingtime of the polymer fiber. The partial pressure of the solvent can alsobe modified via control of temperature, pressure, and mixture ofsolvents. The solvent concentration as determined by a relativeconcentration in the atmosphere is controlled to between 0 and 100%.

Filter Support Structures

In addition to obtaining nanofibers having few defects and a closedistribution in fiber diameter sizes, the construction of a support andpreparation of the surface of the support affect the resultant fiber matand the resultant filter properties. In one embodiment of the invention,a macroscopic mesh provides adequate support for the nanofibers towithstand the forces exerted on filter mat during filtration andcollection of biological medium. The support mesh contributes minimallyto pressure drop of the resultant filter.

Filters formed with rigid meshes that contained 1.27 cm, 0.635 cm, or0.159 cm (i.e., American Engineering standard sizes: ½″, ¼″ and 1/16″respectively) openings using copper, brass, nickel, stainless steel, andaluminum metal are suitable for the invention. Smaller sizes have alsobeen found acceptable including meshes with openings as small as 0.031cm. Aluminum window screen with openings about 1.2 mm×1.6 mm is also anacceptable support. The surface of the metal mesh, especially foraluminum meshes, was subjected to cleaning to remove dirt and oilsfollowed by washing the mesh in diluted sulfuric acid (10 to 20% H₂SO₄in DI water by volume) to remove resistive oxides and impurities. Thiscleaning improved nanofiber dispersion and adhesion. The depositedfibers may not be totally dried of the solvent used to dissolve thepolymers. In that state, the fibers adhere to the rigid mesh and aftertensioning after drying form a mesh-fiber structure beneficial to reducepressure drop and increase collection efficiency. Any number of metalsor metal alloys, with openings of various shapes (square, rectangle,circular, diamond, oblong and odd shaped), with openings ranging in sizefrom about 12.7 mm down to 1000 times the AFD can be used in theinvention.

Adhesion of the nanofibers or fibers to the support mesh can be improvedvia the application of an adhesive to the mesh directly prior toelectrospinning. The adhesive typically is a slow drying adhesivepermitting the adhesive to be tacky (i.e., adhesive) when electrospunfibers are deposited. Alternately, in another embodiment, the wires (orcomponents) of the mesh can be coated with a very thin layer of polymerthat has surface groups which interact (van der Waals, hydrogen-bond,dipole, electrostatic attraction, etc.) with the polymer fibers beingdeposited on the mesh. One example system is a thin coating ofpoly(glycidyl methacrylate) (PGMA) on nickel mesh with nanofibers ofpoly(methyl methacrylate) (PMMA) deposited on the coated mesh. Analternate embodiment of the invention uses cross linkable systems thatare polymerized after the fibers are deposited. Examples includechitosan nanofibers crosslinked with glutaraldehyde and polyvinylacetate crosslinked with borax; also, deposition of nanofibers onadhesives such as Norland's line of curable adhesives based onmercapto-ester compounds. These surface coatings increase adherence andadhesion of the nanofibers to the support.

The metal mesh can be replaced with metal foams such as ERG's Duocel™metal foams; for example, Aluminum Durocel with 20 pores per inch (PPI;alternately an average pore size of 1.27 mm). Foams can also be madewith copper, nickel, and various other metallic as well as polymericmaterials. Porosities ranging from 10 PPI (2.5 mm pores) to 40 PPI(0.064 mm pores) are acceptable for the invention.

The support mesh can be composed of a plastic that is conductive. Forexample polyester or nylon screen (or coarse nonwoven polymer mesh) iscoated with a conductive finish such as gold, palladium, or variousmetal alloys. The coating process can be achieved by any number ofestablished arts including vacuum deposition (e.g., sputter coating,evaporation deposition, and chemical vapor deposition), and chromeplating of plastics. Alternately, the mesh can be composed of conductiveplastic that obtains its conductivity via embedded conductive particles(carbon nanotubes, metals etc.); or, any method to make plastic meshconductive, semi-conductive, or electrostatic dissipating.

A nonwoven support that is conductive or made conductive (e.g., sputtercoating etc., as mentioned above) or moistening with a conductive fluidsuch as water can be used. The nonwoven support can make a largercontribution to the pressure drop but may be acceptable in certainapplications. In certain embodiments, use of woven scrim materials mayalso be acceptable for a base of a bioparticle collection medium.

The structure of the electric fields between the emitter and ground,which drives fiber deposition, are controlled, in part, by the design ofthe filter frame holder. Furthermore, the potential of the support meshcan be controlled by an electric field pulsation device (i.e., a voltagelimiter or discharge device or an electric field applicator device). Theelectric field pulsation device can be configured to pulse an electricfield at the collector at least once (or frequently) duringelectrospinning of the fibers to discharge charge accumulated on theelectrospun fibers.

Electrospun fibers carry charge to the mesh which is dischargedfrequently to ground by the voltage limiter device acting in thisexample as an electric field pulsation device. The resultant electricfield is oriented in the direction of the spinning fibers anddynamically modifies the structure of the electric field, therebyimparting improved fiber and mat properties (as measured by the FoM ofthe mat).

Filters having figures of merit greater than 20 kPa⁻¹ for average fiberdiameters of the nanofibers less than 200 nm and filters having a FoMgreater than 40 kPa⁻¹ for average fiber diameters of the nanofibers lessthan 100 nm have been also realized and are suitable for this invention.

The thickness of the fiber mat can vary from about 0.25 μm (250 nm) to500 μm or beyond if needed, where most filters had an average matthickness in the range of 2 to 5 microns. The average mat thicknessnumbers represent the average thickness of the total fiber mat in afilter. Alternately the mat thickness can be defined as layers of fiberswith the thickness including from 4 to 4000 layers where 4 to 400, or 5to 100, or 5 to 15 layers were typical in various embodiments.

The flexibility of electrospinning even allows mixed polymers such ascoaxial, mixed (blended) in same fiber, or deposited as layered orintermixed fibers. In addition to polymer chemistry and mixture ofpolymers, additives such as salts, proteins, and other materials can beincluded in the fibers via a variety of methods. These include directincorporation in the electrospinning solution, deposited or coated ontothe surface of the fibers during electrospinning using coaxial spinning,electrospin-spray or co-spinning U.S. Pat. No. 7,592,277 (the entirecontents of which are incorporated herein by reference). An alternativeto including additives as a part of the spinning process is to use apost-spinning process to coat the fibrous mat. The fibers can be coatedafter the fibers are formed via depositing the fibers into a liquid bathcontaining the additives or via dry or wet coating of the fiber matafter it is produced. A variety of these combinations is also possible.

Accordingly, the fibers and nanofibers produced by the inventioninclude, but are not limited to, acrylonitrile/butadiene copolymer,cellulose, cellulose acetate, chitosan, collagen, DNA, fibrinogen,fibronectin, nylon, poly(acrylic acid), poly(chloro styrene),poly(dimethyl siloxane), poly(ether imide), poly(ether sulfone),poly(ethyl acrylate), poly(ethyl vinyl acetate), poly(ethyl-co-vinylacetate), poly(ethylene oxide), poly(ethylene terephthalate),poly(lactic acid-co-glycolic acid), poly(methacrylic acid) salt,poly(methyl methacrylate), poly(methyl styrene), poly(styrene sulfonicacid) salt, poly(styrene sulfonyl fluoride),poly(styrene-co-acrylonitrile), poly(styrene-co-butadiene),poly(styrene-co-divinyl benzene), poly(vinyl acetate), poly(vinylalcohol), poly(vinyl chloride), poly(vinylidene fluoride),polyacrylamide, polyacrylonitrile, polyamide, polyaniline,polybenzimidazole, polycaprolactone, polycarbonate,poly(dimethylsiloxane-co-polyethyleneoxide), poly(etheretherketone),polyurethane, polyethyleneimine, polyimide, polyisoprene, polylactide,polypropylene, polystyrene, polysulfone, polyurethane,poly(vinylpyrrolidone), poly(2-hydroxy ethyl methacrylate) (PHEMA),gelatin, proteins, SEBS copolymer, silk (natural or syntheticallyderived), and styrene/isoprene copolymer.

Additionally, polymer blends can also be produced as long as the two ormore polymers are soluble in a common solvent or mixed solvent system. Afew examples would be: poly(vinylidene fluoride)-blend-poly(methylmethacrylate), polystyrene-blend-poly(vinylmethylether), poly(methylmethacrylate)-blend-poly(ethyleneoxide), poly(hydroxypropylmethacrylate)-blend poly(vinylpyrrolidone),poly(hydroxybutyrate)-blend-poly(ethylene oxide), proteinblend-polyethyleneoxide, polylactide-blend-polyvinylpyrrolidone,polystyrene-blend-polyester, polyester-blend-poly(hyroxyethylmethacrylate), poly(ethylene oxide)-blend poly(methyl methacrylate),poly(hydroxystyrene)-blend-poly(ethylene oxide)).

Other embodiments of the invention include the use of polymers that arepH and/or thermal responsive such that the fiber mat can later bemodified, respond to a change in environment, or easily dissolved.Example polymers include the commercial pH sensitive polymers know asEudragit polymers as well as copolymers of N-isopropyl acrylamide(NIPAM) and N-methyacryloy-L-Leucine (MALEU) or (N,N-dimethylamino)ethylmethacrylate (DMAEMA). A similar approach would be to use polymers thatare easily degraded with enzymes such as Chitosan which is degraded byChitosanase and cellulose which is degraded by α-cellulase. Combinationsof polymer systems could be used to tune the fiber filter mat propertiesto the particular application.

Other embodiments of the invention introduce an agent to the fibrousmatrix to reduce oxygen toxicity to bioparticles collected in thecollection medium. Such agents can be enzymes to reduce oxygen toxicityincluding catalase. Such agents can be fullerenes and modifiedfullerenes that have antioxidant properties. In general, substances withknown antioxidant properties can be added to the sample collectionmedium of the present invention to improve viability of the collectedbioparticles.

Accordingly, in one embodiment of the invention, fibrous mats can beelectrospun that provide favorable conditions for a variety oforganisms.

Working Examples of Fibrous Mats for Bioparticle Collection

A variety of polymer fiber mats were prepared for this invention usingelectrospinning. The polymer solution was prepared to a targetconcentration in a solvent system amenable to electrospinning. Thesolution was placed in a 3 ml syringe fitted with a 30 G blunt tippedneedle and placed into a controlled gas environment (see U.S. Pat. No.7,297,305, the entire contents of which are incorporated herein byreference) of carbon dioxide and a relative humidity (RH) of 30% to 35%.A high voltage was provided to the needle through an electrical lead tocreate a voltage gradient of about 1 kV/cm. The polymer flow rate wasdetermined by the electrospinning solution viscosity and ranged between0.05 to 0.1 ml/hr. The fibers are collected on a substrate dependingupon the manner in which the fiber mat was used.

For use as an impaction substrate, the collection substrate was aluminumfoil or Whatman filter paper; for use as a flow-though filter, thecollection substrate was a light weight nonwoven filter material such asFiberweb Reemay style 2011, which is a nonwoven filter material with abasis weight of 25.5 g/m² and an air permeability of 5,650 L/m²/s. Insome cases, the nonwoven backing is first coated with graphite, such asby spray painting using Aerosdag G to enhance fiber adhesion to thesubstrate. The electrospun fiber mat was then rinsed with filtered DIwater and dried in a sterile environment overnight to remove anyresidual solvents. In some cases, additives were coated onto thefinished mat in a post-processing step.

One fibrous material prepared was polysulfone (PSu; Udel P3500 LCD bySolvay Advanced Polymers) dissolved in dimethylacetimide (DMAc) at aconcentration of 21 wt %. 0.2 wt % tert-butyl ammonium chloride (TBAC)was then added to improve the electrospinning of the solution and thefinal fiber morphology. The solution was electrospun for 90 minutes at aflow rate of 0.05 ml/hr and voltage gradient of 1.6 kV/cm.

Another fibrous material prepared was polyurethane (PU; Pellethane byLubrizol) dissolved in dimethlyformamide (DMF) at 13 wt %. The solutionwas electrospun at 1.2 kV/cm and a flow rate of 0.1 ml/hr for 90minutes. More specifically, Pellethane 2103-90 AE Nat polyurethane (PU)made by Lubrizol, electrospun at about 13 wt % in dimethlyformamide(DMF) to form micro and/or nanofibers was deposited on a backingmaterial. After the fibers are deposited on the backing, the fibrousmatrix was flushed with DI water and allowed to dry in a cleanenvironment.

The backing material can be any number of woven or nonwoven media suchas spunbound polypropylene. One example is Reemy spunbound polypropylenenonwoven made by Fiberweb. Media with air resistance of 500 CFM/ft2 to1,500 CFM/ft2 are useful but media with air resistance beyond this rangemay also be useful. In some cases, using a backing material that isconductive or static dissipating is advantageous. For example, anonwoven can be spray coated with graphite or coated with conductivematerial using liquid or gas based (e.g. chemical vapor deposition)techniques. Also materials known in the art that are static dissipatingthough any number of methods may be useful.

Another example of a nanofiber structure for viable collection andpreservation is PU electrospun onto Fiberweb Reemy 2250 that was coatedwith aerodag (graphite) before electrospinning. The PU fibers have anaverage fiber diameter of 320 nm, are free of beads, and form a nonwovenmat that is a few microns to 10 s of microns thick. Alternatively, thePU fibers can have an average fiber diameter in the range of 100 nm to280 nm. Alternatively, the fibrous matrix can have PU beads createdduring the electrospinning process that are 1.5 to 4.5 microns indiameter. In one embodiment, the average fiber diameter is 260 nm, andthe PU beads are about 4 microns in diameter. These nanofiber materialswith beads about 10 times to 20 times the size of the fibers providelower pressure drop.

FIGS. 10 and 11A-11C show SEMs of one embodiment of the nanofiberstructure formed by deposition of PU on Fiberweb that was first coatedwith graphite. In FIG. 10, a section of backing material withoutnanofibers and a section with nanofibers are shown. The nanofibers aresupported by the nonwoven backing material. The nanofibers provide forcollection of the bioparticles. In FIG. 11A, the beaded structure of thenanofibers and that they oriented between the supporting macrofibers ofthe conductive nonwoven they are deposited on is evident. Thiscombination of beading and fiber orientation provides a structure thathas lower pressure drop compared to other materials. In FIG. 11B, anedge (cross sectional) view of the nanofibers deposited upon thenonwoven backing is shown. The nanofiber layer is 10 s to 100 s ofmicrons thick, but much thinner than the supporting nonwoven material.In FIG. 11C a high magnification SEM image is shown that indicates thefiber and bead structure. The beads are typically oblong and are 10 to20 times the size of the fibers. The fibers are on the order of 220 to280 nm while the beads are on the order of 3.8 to 4.8 microns.

Another fibrous matrix of this invention includes nylon fibers preparedfor example from a 15.3 wt % solution of nylon 6 (Sigma Aldrich)dissolved in formic acid. The solution was electrospun at a voltagegradient of 1.6 kV/cm and a flow rate of 0.05 ml/hr for 90 minutes.

Another fibrous matrix of this invention includes polycaprolactone (PCL;Sigma Aldrich, ca 43,000 M_(w)) fibers prepared from a mixed solventsystem. The solvent was composed of 80% methylene chloride and 20% DMF.PCL was dissolved in the mixed solvent to a concentration of 18 wt % andelectrospun at 1.1 kV/cm with a flow rate of 0.1 ml/hr for 90 minutes.

Another fibrous matrix of this invention includes polystyrene (PS; SigmaAldrich, ca 350,000 M_(w)) fibers prepared from 23 wt % PS in DMF andelectrospun for example at 1.2 kV/cm and a flow rate of 0.1 ml/hr for 90minutes.

In one embodiment, a fiber-facilitator is used. With a fiber-facilitator(e.g. high molecular weight PEO), nanocellulose and related cellulosicmaterials can be incorporated in the fibrous matrix to form fibers. Inother words, to assist in incorporating cellulose-based materials intofibers, a facilitator which is known in the art (e.g., high molecularweight PEO) to help in the formation of fibers, can be used.

One way according to this invention to impart improved viabilitymaintenance to the fibrous mat was to apply a solution containingadditives to the electrospun mat after it was made and rinsed with DIwater. The coated mat would then be allowed to dry in a sterileenvironment. For example, a solution containing a protein containingsolution tryptic soy broth can be applied to the mat and allowed to drybefore use.

Working Examples of Flow-Through Filters for Collecting Bioparticles

A sheet of nonwoven nanofiber filter media was prepared using PSu asdescribed above with the fibers being deposited on graphite coatedfiberweb support material. 37-mm circle filters were punched out of thesheet of nanofiber filter media and packaged into a standard 37-mm airsampling cassettes for testing.

FIG. 11D shows a nanofiber sampling filter in the 37 mm cassette formatrealized by this invention. The efficiency of the filter cassette wasmeasured with 300 nm KCl aerosol particles and was found to be >99.9%and have a pressure drop of 167 Pa for a face velocity of 5.3 cm/s. Asimilar filter cassette was exposed to bioaerosol, Bacillus globigii(Bg) or MS2, and analyzed via SEM and molecular biology techniques.Collection of Bg spores and MS2 particles was obtained. FIG. 12A showsSEM images of a collected Bg spore and what are likely MS2 virusparticles.

In one embodiment of the invention, the collection efficiency of thefiber mat structure (sampling filter) formed from a plurality of microor nanofibers can be >80% and more specifically >95% for particles 0.025μm to 10 μm in diameter for a flow rate of 25 L/min for a 25 mm samplingcassette. While at the same time, the pressure drop (air resistance) ofan unloaded fibrous sampling filter is less than 20 inches of water, andmore specifically less than 12 inches of water.

FIG. 12B compares the pressure drop curves (pressure drop versus facevelocity) for two common commercial air sampling filter materials withnanofiber filter media composed of PSU or PU deposited on graphitecoated fiberweb as described above. The structure formed by thelightweight backing material, the small fiber diameter, the partiallyoriented fibers, and beaded fibers (exemplified by PU nanofibers)provides for significant reduction in pressure drop. These significantlylower pressure drops of the nanofiber filters translate into advantagesfor both operation and equipment design. With a lower pressure dropacross the filter it is easier to maintain the target RH of the filterand therefore improve viability maintenance of the collectedbioparticles. Furthermore, with lower pressure drop the pumps andelectrical requirements for an air sampling device are smaller and morecost effective.

Remarkably, despite the efficiency >95% and pressure drop less than 12inches of water, the sampling filter is able to withstand loading withparticles until pressure drops greater than 80 inches of water, and evenas high as 100 inches of water.

The fibrous sampling filter described above is able to operate atcollection humidities ranging from 10% to 98% with no loss of filteringintegrity. However for viability considerations, it is preferablyoperated in the range of 70% to 85%.

While described here in relation to flow through sampling, these fibroussampling filters have application in the other sample collection devicesdescribed herein.

Bioparticle Collection, Testing, and Evaluation

Viable microorganisms were generated to test the viable collection ofthe samplers. Bioparticle generation was accomplished though the use ofa Collison nebulizer containing a suspension of microorganism. Themicroorganisms may be suspended in various nebulizing fluids dependingupon the organisms and the scenario being tested. Nebulizer fluids rangefrom sterile water to tryptic soy broth with antifoam. The compositionof the nebulizing fluids is often selected to simulate the conditions ofvarious bioparticles in the environment as the usual application forbioparticle samplers is to collect microorganism from the ambient orindoor air.

A recognized standard in the art of bioparticle collection is the AllGlass Impinger (AGI). The AGI is designed to draw aerosols through aninlet tube (e.g., a capillary tube) to form a jet of the aerosols to becaptured by a liquid medium of deionized water or impinger fluid. Thejet tip is typically positioned 30-mm above the base of the impinger.The AGI relies on the inertial impaction as a means for collection.However, loss of sampling liquid through evaporation andre-aerosolization of droplets containing virus often reduces collectionefficiency of liquid impingers.

The AGI provides collection into liquid for particles larger than about0.3 microns. Due to the wet collection, the majority of sampledorganisms are collected in a viable state. However, this method can onlybe operated for a short period of time, about 30 minutes. Yet, the AGIis a recognized collection system used as a point of comparison to thefibrous material collection devices of this invention. Andersenbiological impactors and the SKC biosampler were also suitable. Theliquid collection fluid is diluted (when necessary) and then analyzed.

Sampling of a controlled air stream containing an aerosol of a microbeat controlled concentration was conducted to compare sampling methods.In some cases an AGI is run for 30 minutes in parallel with the othersampling technology with the AGI being considered the “gold standard” tocompare viable sampling collection against. For example for a specifiedtest bacteria, 6% of AGI means that the method collected 6% of thecolony forming units per liter of sampled air that the AGI collected

Meanwhile, the fibrous material collection devices of this inventionwere suspended in sterile extraction fluid (e.g—water, phosphatebuffered saline, tryptic soy broth [TSB]), diluted and analyzed.

For comparison, the analysis for culturable organisms followed standardprocedures where an aliquot of collection fluid, extraction fluid, or adilution of either, is plated on microbiological media appropriate forthe microorganisms collected. The plated media were incubated at atemperature favorable for the microorganism growth and enumerated whencolonies (bacteria) or plaques (viruses) are countable.

Evaluation of Collection Via Filtration and Humidity ControlledFiltration

The methods sampling of air using filtration and of adding moisture orcontrolling the humidity of the sampled air followed by filtration wereevaluated using aerosols of bioparticles and comparison with industrystandard filtration sampling methods or the AGI.

FIG. 13 includes Table 2 showing a comparison sampling the virus MS2using filtration without humidity control of the sampled air. Thenanofiber filter mats of the invention are compared to a standard Teflonfilter. Table 5 assesses both collection efficiency and viability. Thecollection efficiency of the polysulfone-based nanofiber filter wasnoticeably higher than the standard Teflon filter or the polystyrenenanofiber filter. (These results are for materials not optimized for aspecific microbe collection.)

For demonstration of humidity controlled filtration, a method ofcontrolling the humidity of the sampled air was constructed thatmeasured the RH immediately downstream of the sampling filter. The RH ofthe sampled air was controlled via mixing with a moist air stream,similar to that shown in FIG. 4. The moist airstream was generated bypassing clean, dry air through a bubbler containing deionized waterfollowed by HEPA filtration of the humidified air. The ratio of sampledair to wet air was set at the beginning of the experiment to providetarget RH at the filter. This ratio was noted and used to determine theactual volume of air with aerosol sampled from the test chambercontaining the aerosolized bioparticles. Various filter types includingnanofiber filters of this invention were used. In the art, gelatinfilters are recognized as having the best viable collection ofcommercially available materials. However, when used on their own, as istypical in the art, they too are very limited in the duration time forviable sampling of bioparticles, about 30 to 60 minutes.

Humidity controlled filtration was performed with the vegetativebacterium Serratia using nanofiber filters of this invention compared togelatin or Teflon filters. The nanofiber mats were punched into 25-mmcircular filters and placed into a standard 25-mm air sampling cassette.The gelatin and Teflon filters were used as received in a 37-mm airsampling cassette. Bioaerosol was sampled for 3 hours and the results ofCFUs of Serratia determined. FIG. 14 compares the results of thisexperiment. The nanofibers and gelatin perform better than the Teflonfilters. It should be noted that the gelatin filters have much higherpressure drop than the nanofiber filters, see FIG. 12B.

A similar experiment to that shown in FIG. 12B was attempted with longerterm sampling to compare gelatin and PU nanofiber filters for very longsampling times. However, the gelatin filters deteriorated sometime after3 hours of sampling and collected bioparticles could not be recovered.The PU nanofiber filters were found to withstand sampling times of morethan 32 hours of operation.

The impact of sampling face velocity as a function of filter materialfor RH controlled filtration was tested with 30 minute sampling ofSerratia as shown in FIG. 15. PU nanofibers and gelatin filters wereused with an RH of 75% and the face velocity varied. The PU nanofiberfilter is able to collect viable bioparticles at very high facevelocities that actually result in rupture of the gelatin filter. Asexpected the percent viable collected increases with face velocity asmore organism per area of filter are collected at the higher flow rates.Operation at high flow rates, as high as 100 L/min or even higher, isdesirable for air monitoring. For example release of biological weaponcould result in low concentrations of the organism in the air such thatsampling as much air as possible to generate as much collected organismas possible is desired (that is as much single collected for the eventas possible).

To demonstrate collection of a very fragile vegetative bacterium that isparticularly difficult to collect in viable form using filtration,Yersinia rohdei was collected using RH controlled filtration with PUnanofiber filters compared to an AGI. FIG. 16 shows that indeedcollection of even this very fragile organism is possible andrepeatable.

RH controlled filtration using nanofibers is an effective way to performlong term viable collection of bioparticles. Using the fibrous materialcollection devices of this invention provides viable collection similarto gelatin when both substrates are used in the same sampling system andsame RH for short periods of time and modest flow rates (face velocityless than 4,500 cm/min). However, the fibrous material collectiondevices of this invention provide several advantages over gelatinfilters: 1) filter pressure drop for nanofibers is much lower for thefibrous material collection devices than gelatin; 2) the robustness ofnanofibers in the fibrous material collection devices is much greaterthan gelatin. The fibrous material collection devices are able towithstand high pressure drops, in excess of 100 inch-H₂O, are able towithstand long term operation at >75% RH (e.g., more than 3 hrs, morethan 24 hrs). Furthermore, the nanofiber filters are free ofcontaminants that would interfere with or give false results formicrobiology assays.

Storage of Collected Viable Bioparticles

After collection, preserving the organisms is a significant challenge,particularly in the case where samples are not refrigerated.

In one embodiment of the invention, there is provided an automatedsystem for sequential particle collection and storage. In one embodimentof the invention, several days of samples are stored in a singlesampling cassette that will also contain an electronic tag indicatingindividual sample collection times, location, air sampling volumes, andquality assurance (QA) parameters (e.g., flows, operating temperatures,water levels, and other performance parameters). Another cassette caninclude consumables, including water for the CGT operation or provisionof elevated RH by other method and any supplies for sample collectionand preservation, depending upon the collection and handling schemeselected. In one embodiment, these cassettes would be exchanged in thefield during operation of the sample collector.

As a demonstration, organisms were inoculated via pipetting solutioncontaining microbes onto samples of various nanofiber or fibersubstrates and other substrates compatible with air samplers. Two typesof inoculations were done to simulate different environmentalconditions: “lightly protected” where a buffer with 0.25% TSB in sterilewater was used, and “well protected” where a full strength TSB bufferwas used. When an aerosol containing microorganisms or otherbioparticles is generated in the natural world, it always has othermaterials with it such as proteins, sugars, sputum, dirt etc thatprovides protection for the organisms. In a bioterrorist act, thebioparticles would be purposely mixed with protective materials likeprotein. The samples in this demonstration were stored at ambienttemperature (approximately 23° C.) in controlled RH static chambertests. The relative humidity RH in this demonstration was controlled viasaturated salt solutions in the sealed chambers. After storage, sampleswere extracted in buffer, such as TSB or phosphate buffered saline, andplatted on appropriate nutrient media and incubated and organismsenumerated.

FIGS. 17A and 17B include Tables 3 and 4 showing the results ofsurvivability of model organism on various materials. “Alive” means theorganism was detected via live culture techniques and “dead” means nonewere detected. A “D” and number indicated by days of live detection,e.g. D7 means live culture detected at day 7.

In another set of experiments where PU nanofiber filters of thisinvention were further studied, model organisms were inoculated viapipetting and samples stored under various conditions as shown in FIG.18. The log change from the day of inoculation (day 0) is reported.Storage of the slightly fragile Staphylococcus is possible under avariety of conditions. The very fragile organism Yersinia requiresstorage at cooled temperatures such as 4° C.

Storage of a range of bioparticles on nanofibers is possible. Fororganisms that are hardy to moderately hardy, storage under conditionsnot requiring cooling is possible. In some cases storage under humiditycontrolled conditions such as those provided by the RH control system ofthe sampled air are sufficient to preserve the collected bioparticles.In the case of fragile and very fragile organisms, cooled storage isrequired if viability maintenance for more than a day or two arerequired. As demonstrated, different collection substrates provideviable maintenance for different organisms. With the flexibility ofelectro spinning and other arts of making fibrous media a mixed polymerfiber environment can be created to provide for viable storage of abroad range of organisms not possible with a single traditionalmaterial.

Prior to the invention, the best filter for collection viability thatcurrently existed was a gelatin filter. However, the gelatin filter hasa number of problems including contamination and excessive drying duringlong term sampling without RH control, which both negatively impact thestorage viability. Another common filter medium is PTFE (Teflon) filter.Yet, the results above, especially for the design limiting organisms,show that both collection and storage viability and the collectionefficiency are enhanced for the fibrous material collection devices ofthis invention.

The above experiments with aerosolized bioparticles demonstrated thatnanofibers are good collectors of microbes (bioparticles). The aboveexperiments show that the selection of polymer and fiber structure isone element impacting viability and controllable by this invention. Theabove experiments show that preventing desiccation of bioparticles isimportant is one element impacting viability and controllable by thisinvention. The above experiments show that viability maintenance can beachieved through incorporation of viability sustaining additives,moisture, etc. and by keeping the fibrous media in an RH regulatedenvironment. These aspects are controllable by this invention.

Alternative Applications

In addition to the collection of bioparticles, the various embodimentsof the sampler can collect other aerosol particles of interest to thepublic health and air monitoring communities. The sampler may be usedoutdoors to sample ambient air or for sampling indoors in buildings,arenas, or transportation facilities. These filters also offer anadvantage because of their semi-transparency for black carbon absorptionanalysis and low levels of analysis interfering metals.

In these applications, the ambient air samples will contain black carbonor soot from combustion sources, industrial pollution, particles fromatmospheric reactions, particles re-suspended from soil and pavements,ocean generated particles and pollen, all of which can be collected bythe nanofiber collection devices of this invention. When used for indoorapplications, it is expected that the occupant generated particles suchas skin cells and residue of personal care products, dust and fibersresuspended from carpets and floors, smoking, and particles introducedfrom appliances such as electrical motors and heaters or furnaces, andbiological material such as toxins and plant or animal debris, all ofwhich can be collected by the nanofiber collection devices of thisinvention. Aerosol particles collected in the nanofiber filters could bemeasured by light absorption or reflectance, microscopy, weighing andchemical analysis.

While described above with respect to aerosol sampling, the nanofibermedia and aspects contributing to viable collection and maintenance ofbioaerosols have applications in the sampling of bioparticles andorganisms from surfaces and from water. For example, a wipe or brush orother sample collection device containing the nano or microfibermaterial described above that provides sample collection and helpsviability maintenance could be used to collect bioparticles from akeyboard, lab bench, furniture, vehicle interior, etc. The wipe or brushor other collection device can then be transported with the viabilitymaintenance materials to a laboratory for analysis.

In one embodiment of the invention, the sample collection device can bein the form of wipes, brushes, swabs, sorbent pads, liquid filters, airfilters, and/or similar devices for sampling air, liquids, or surfaces.Applications include forensics, regulatory compliance, surveillance,etc.

For embodiments of the invention where the nanofiber material is used tocollect microbes without incorporation of mechanisms to control thehumidity of inlet air, a container can be used that provides a humiditycontrolled environment. For example a wipe composed of a plurality offibers with viability enhancing properties for use in evidencecollection. In one embodiment that nanofiber wipe is stored in a sterilecontainer with humidity regulation. In other embodiments the wipe isstored sterile but humidity is not required prior to use. The nanofiberwipe is then used to collect a sample and is placed in the containerwhere the container provides a favorable RH environment for viabilitymaintenance during transport to the laboratory for analysis. Thecontainer and wipe thus constitute a sample collection device thatprovides for viability maintenance.

The samples can then be stored in a sample storage device which canincorporate a moisture providing material or mechanism such as ahydrogel, water saturated salt solution, water reservoir separated fromthe nanofibers via a moisture permeable membrane providing watertransport, or a water reservoir connected to the part of the containerholding the nanofibers via a wick. Examples of these are shown in FIGS.19 and 20. FIG. 19 is a schematic depiction of a sample storage deviceincorporating a moisture providing material. FIG. 20 is a schematicdepiction of a sample storage device incorporating a moisture providingmechanism (e.g., a wick and a water reservoir). The moisture providingmaterial and the moisture providing mechanism can further providenutrient or antioxidants, as described above.

Local Environmental Control

One aspect of maintaining the viability of a collected bioparticle iscontrol of the humidity and temperature conditions of capture andstorage. During testing under room temperature conditions, it becameclear that various more fragile organisms required cooler than roomtemperature conditions for storage to maintain viability. Such fragileorganisms include but are not limited to Pox viruses, Filoviruses,Arenaviruses, Alphavirus, Brucella species, Burkholderia mallei,Yersinia pestis and Coxiella burnetii.

During a 24 hour sampling period at room temperature and controlledhumidity, these organisms when collected in the early part of the periodmay not survive the full sampling time. These organisms were found tohave acceptable recovery for shorter sampler times. In addition, theinventors have found that storage of these fragile organisms on ananofiber collection medium as described above at room temperature foreven a 24 hour sampling resulted in substantial microorganism death.

Yet, the ability to collect and store the samples on a nanofibercollection medium at controlled, refrigerator-type temperatures, andfrom wide variety of ambient (and changing) conditions in theatmospheric (ranging from humid summers, dry summers and cold winters)represents a formidable challenge. To illustrate this point, FIG. 21depicts a scatter plot of metrological year data showing the range oftemperature and relative humidity conditions from diverse locationclimate conditions in the United States of Phoenix, Ariz.; Duluth,Minn.; Washington D.C. and Denver, Colo. While shown for geographicalpoints in the United States, the same challenges occur in samplingaround the world.

Described below are techniques and equipment suitable for this inventionto promote capture and storage of bioparticles in a local environment(i.e., a contained environment about the collection media) in which thelocal environment has both below room temperature control and humiditycontrol. In particular, while the invention is not limited to exactly atemperature of 4° C., a temperature condition of 4° C. has been found tobe suitable and useful for both sampling and storage of the collectedsamples of microorganisms. The description below often refers to the 4°C. condition, but other temperatures and ranges of temperature could beused by the invention dependent for example on the particularmicroorganism being collected. Suitable temperature ranges for thisinvention include 2° C. to 6° C. conditions, 1° C. to 7° C. conditions,4° C. to 10° C. conditions.

FIG. 22 is a psychrometric chart showing potential ways of conditioningthe bioaerosol sample to the ideal sampling and preservation conditionsat 4° C. and thereby illustrating the relevance of metrologicalconditions in reaching sampling conditions. The area outlined by thechart in FIG. 22 shows the range of ambient conditions expected forambient sampling. The rectangle (in the lower left) at 4° C. is thetemperature of a typical refrigerator and represents one of the targetenvironmental conditions of this invention for sampling and storage ofbioaerosol samples. The challenge is to transport and condition abioaerosol sample without killing the organisms and while not crossingthe saturation line of the chart to prevent wetting of the collectionmedium and to prevent “washing” of the microorganisms out of the gasstream.

FIG. 23 is a psychrometric chart illustrating preferred paths forconditioning the bioaerosol sample to avoid killing the organisms. Onepreferred path according to one embodiment of the invention would be apath through temperature and humidity space where the survivability ofthe organisms is not endangered by overheating or overdrying of theorganisms. One such path shown in FIG. 23 is along a contour of relativehumidity permitting simultaneous heat and moisture transfer to avoidcondensation or extreme dryness. The curved path can be replicatedthrough a “staircase” series replicating this control path. A verticalline is shown to depict a path for achieved with adjustment of theabsolute water vapor pressure at constant temperature, as done with theapparatus of FIGS. 24A and 25A. Similarly, if one needs to raise eitherthe temperature or the dew point to achieve the target sampletemperature and dew point, this can be done by adjusting first the dewpoint and then the temperature so as not to cross over the vaporsaturation line. At a constant temperature the dew point, or relativehumidity, can be adjusted to a target value using a (e.g., Nafion™-type)device as shown in the apparatus of FIGS. 24B and 25C. Options such asinjecting water sprays for cooling are undesirable because of possiblescrubbing and removal of the bioaerosol and contamination from materialsin the water. Also, direct contacts with chemical drying materials alsohave similar problems with contamination.

Also, direct contacts with chemical drying materials also have similarproblems with contamination. A vertical line is shown to depict a pathfor drying with a conventional (e.g., Nafion™-type) device becausedehumidification in a conventional device is conducted under a nearlyconstant temperature.

The invention provides in one embodiment an aerosol collection systemincluding a bio-aerosol delivery device configured to supplybioparticles in a gas stream, a moisture exchange device including apartition member coupled to the gas stream and configured to humidify ordehumidify the bioparticles in the gas stream with a vapor of abiocompatible liquid, and an aerosol collection medium downstream fromthe moisture exchange device and configured to collect the bioparticles.The moisture exchange devices or units of this invention can include atleast one of a permeable material, a semi-permeable membrane material,or a polymeric ionomer.

The addition of a Nafion-type moisture exchanger as shown in FIG. 26provides for control of relative humidity for bioaerosol sampling whichcan be controlled in the manner noted above. FIG. 26 illustrates aschematic of a common way of implementing moisture exchange asmanufactured by the Perma Pure LLC company. The Nafion is formed intosmall diameter tubes. These small diameter tubes are sealed into amanifold top and bottom to allow of treatment bioaerosol through thedevice. The small diameter tubes are mounted as a bundle in a largertube with side penetrations to allow the flow of moisture containing airfor exchange with the bioaerosol within the tubes.

The illustration of the inlet and outlet plenums shown in FIG. 26 showsthe principle that bioaerosol entering through a single tube must betransported to a multitude of small diameter tubes for rapid moistureexchange. At the exhaust of the tubes, the flows must be recombined fortransport through a single tube. Bioaerosol are composed of discreteparticles generally in the 0.1 to 10 micrometer particle size range.

The moisture exchange units used in this invention (such as theNafion™-type units noted above) have been designed for minimumturbulence in the plenums by eliminating abrupt expansions andcontractions of air flow and provide for aerodynamic entry into thetubes by increasing the radius of curvature of the tube inlets to reduceloss of bioaerosol particles. Another way of collecting bioaerosol couldutilize tubular membrane moisture exchanger as well.

In FIG. 27, the principle of controlling both the relative humidity andtemperature is illustrated. A temperature controlled environment is usedto contain the bioaerosol sampling device such as nanofiber filters. Thetemperature controlled environment may also contain the tubular membranemoisture exchanger as well.

According to one embodiment of the invention, as noted above, thepreferred conditioning path would not penetrate the dew point and formliquid water. The inventors have discovered a controlled path existingbetween excessively low relative humidity avoiding organism desiccationand excessively high relative humidity leading to water condensation.

FIG. 24A is a schematic diagram depicting a bioaerosol sampling systemof this invention. The bioaerosol enters the top of the system. Aheater/cooler unit is need for either hot or cold conditions.Potentially, this heat exchanger could be a liquid circulation systemusing the refrigerator than the thermal source. The bioaerosol nextenters Nafion™ gas phase water exchanger. FIG. 24B is a schematicdiagram depicting the principle that multiple moisture exchange(humidifier/dryer) devices can be employed in series and can be locatedin the temperature controlled space.

Nafion™ (a polymeric ionomer) is a copolymer ofperfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid andtetrafluoroethylene (Teflon®) in a class of materials called polymericionomers. In simpler terms, Nafion is a Teflon backbone with occasionalside chains added of another fluorocarbon. The side chain terminates ina sulfonic acid (—SO₃H). With the use of Nafion in dryers or humidifier,the Nafion functions essentially as a highly selective, semi-permeablemembrane to water vapor. If gases inside the Nafion tubing are wetterthan gases surrounding the tubing, drying will occur. If the surroundinggases are wetter, humidification will occur.

In this invention, a bioaerosol sample flows through the inside of thesemi-permeable membrane tube side while a conditioning gas flows on theshell side. The amount and direction of water transport through thesemi-permeable membrane wall depends on the water gradient between tubesand shell. Multiple, moisture exchangers could be used in a cascadedseries as shown in FIG. 31. FIG. 33 illustrates the cascading ofconditioning steps in a concept to reduce the loss of bioparticles tothe wall from excessive expansion and contraction from the flowingbioaerosol. Other semi-permeable polymers beside Nafion can be used.Suitable water vapor-permeable materials for this invention includepolymer nonelectrolytes (such as polyethylene oxide, polyvinyl alcohol,cellulose ether and starch) and copolymers thereof; polymer electrolytes(such as polyacrylic acid, polyacrylamide, polyisopropyl acrylamide,polystyrene sulfonic acid, polyvinyl pyridine and polyamino acid) andcopolymers and salts thereof; and conventional water-absorbing resinsand conventional water-containing resins. Also, it is possible that flatsheet membranes can be used in some applications rather than tubes. Inthose applications, the bioaerosol particles would be partitioned offfrom the humidified or dried air by at least one wall of thesemi-permeable membrane.

In one embodiment of the invention, a bioaerosol sampling filter is heldin the refrigerator section of the bioaerosol sampling system. Thesampling filter (as described above) can be made of nanofiber materialto improve perseveration and allow sampling at a high face velocity.Also, multiple filter holders with a common inlet plenum and controlledwith a multi-port valve to allow obtaining single day samples overseveral days can be used. The refrigerator section of the bioaerosolsampling system can also be used as a source of cold dry air. Anexternal refrigerator or dehumidifier (not shown) could also be used asa source of cold dry air.

The control system depicted as a box in FIGS. 24A and 24B regulates thewater vapor removed or added by controlling the water vapor content inthe shell side flow of the Nafion water exchanger. A condenser is shownto indicate that removal of water from the shell side flow from theNafion water exchanger might be accomplished by cooling. Multi-daysampling might be accomplished by the use of multiple sample holderswith flow switched with a multiport valve or by automated filterchanging. It is expected that these schemes would be contained withinthe refrigerator section of the bioaerosol sampling system.

Control of the relative humidity at the point of sampling at ambienttemperature can be accomplished with the system of FIG. 25A. One key isto either humidify, or dehumidify, as needed to bring the sample flow RHto 75% to 85% upstream of the point of collection. FIG. 25A is adepiction of a humidity control system of this invention. The specificsystem pictured in FIG. 25A adjusts the RH to the target RH at thesample temperature. Regulation of the total pressure of the humidifiedair flow in the shell side of the Nafion system provides a mechanism foradjusting the relative humidity of the sample flow. The shell siderefers to the interstial space between the Nafion tubes that carry thesample flow. Typically, the shell side flow rate carries a counterflowat a mass flow rate equal to about twice the sample flow. ThisNafion-based relative humidity conditioner adjusts the relative humidityof the sample stream to the target relative humidity, either humidifyingor dehumidifying as needed, by controlling the absolute pressure ofwater vapor in the shell side. This absolute pressure is controlled byadjusting a valve immediately upstream of the inlet to the shell. Theflow through this valve creates the pressure drop, and thereby controlsthe absolute pressure of water vapor in the shell side. The system inFIG. 25A in one embodiment is designed to humidify air to 70% RH at aconstant temperature. The system either humidifies or dehumidifies bycontrolling the pressure of highly saturated air flow in the shellregion. When the shell pressure is near atmospheric pressure, the dewpoint of the shell flow is also high and this brings the RH of thesample flow to a high relative humidity. When the pressure of the highlysaturated air flow in the shell is at a reduced pressure, perhaps as lowas one-half of an atmosphere, the dew point is likewise reduced becausethe absolute pressure of water vapor is reduced along with the totalpressure. If the shell dew point is lower than the sample dew point thesystem Nafion system will dehumidify. The control of the pressure in theshell region, and hence the RH achieved, can be controlled automaticallythrough the adjustment of the valve based on the reading of the RH atthe sample temperature. Thus both functions of humidification ordehumidification are incorporated in the same apparatus.

In one embodiment of this invention, the membrane-based moistureexchange devices are tube in shell devices. This geometry creates abarrier between the bioaerosol and the conditioning air preventingcontamination. Conventionally, Nafion™ systems are used at constanttemperature. In the present invention, if cold air at 4° C. wereintroduced at the bottom port on a Nafion-type moisture exchanger inFIG. 25B, then a temperature gradient would exist up the axis of thedevice. Although not a primary consideration for bioaerosolconditioning, reducing the temperature of purge gas or jacket flow hasthe additional advantage of improving the ability to reduce the dewpoint of the conditioned gas.

Control of the relative humidity and temperature at the point ofsampling, where the temperature at the point of sampling is between 1°C. and °10 C., requires conditioning the sample air flow to along apathway on the psychometric chart of FIG. 22 such that the sample flowremains subsaturated at all times, preferably in the relative humidityrange between 40% and 90% RH. This combined temperature and relativehumidity conditioning can be accomplished with the two-stage systemillustrated in FIG. 24B (and FIG. 25C). Potentially the controlledprocess line on FIG. 23 could be realized because both the temperatureand relative humidity would be changed in the membrane-based moistureexchange device of this invention. Examples of the potential membranebased devices are shown in FIGS. 28, 29, and 30. The gradients shown inFIGS. 28, 29, and 30 are merely representative of the temperature andhumidity gradients used in the invention. As can be noticed, the maximumtemperatures typically conform to outside ambient temperatures. As canbe noticed, the predetermined temperatures typically conform to thetargeted 4° C. type conditions. The same applies for the RH gradients.The length of the tubes in FIGS. 28, 29, and 30 over which the gradientexists is typically 15 cm, but distances as short as 1 to 2 cm and aslong as 100 to 1000 cm (or possible greater) can be used. In thesearrangements shown in FIGS. 28, 29 and 30, the properties of combinedmoisture and heat transfer are used to advantageously improve theviability of the collected microorganisms.

The driving force for moisture transfer is the difference in water vaporpressure between the bioaerosol in the tubes and the air introduced inthe shell side of the device. The dew point is the temperature ofmoisture saturation. The relative humidity critical for bioaerosolviability is the ratio of mole fraction of water vapor of the bioaerosoldivided by the mole fraction at saturation. Therefore, a path may beselected to maintain relative humidity with changing temperature and dewpoint temperature such as illustrated in FIG. 28.

Arrangements in FIGS. 29 and 30 might be used with advantage in acascade as shown in FIG. 31. The vapor pressure of water (P) as afunction of absolute temperature (T) in equilibrium with sorption sitesin this device is given by (K. J. Leckrone and J. M. Myers, “Efficiencyand temperature dependence of water removal by membrane dryers” Anal.Chem, 1997, 69(5), 911-918.)Log P=−3580/T+10.01

The implication is that the membrane-based moisture exchange device whenapplied as a dryer becomes much more effective as the temperature isreduced. Also the opposite is true, that elevated temperatures wouldfacilitate humidification of the sample air. Further, heat transferbetween the shell flow and tube flow would depend on the temperaturedifference across the membranes. However, the condensation of water isavoided by the control system of this invention to prevent loss ofbioaerosol and reduction of efficiency of the moisture exchanger.

Factors which impact the collection and storage of viable microorganismsdepends on a number of variables:

-   -   Type of membrane in membrane-based moisture exchange device    -   Diameter and wall thickness of the membrane tubes    -   Number of membrane tubes    -   Length of membrane tubes    -   Plenum design in the moisture exchangers (The plenum design and        the tube diameter will affect bioaerosol losses in the device.)    -   Air flow rate of both the tube flow and shell flow. Normally the        shell flow rate is at or above the tube flow rate.    -   Temperature and relative humidity of the entering tube flow and        entering shell flow.    -   If a concurrent flow or counter current flow scheme is used        between tube flow and shell flow.

In this invention, a series of moisture exchangers could be employed.Examples include the following:

-   -   Cold dry sample—hot wet co-current shell flow to prevent drying,    -   Hot dry sample—cold wet concurrent shell flow to prevent drying,    -   Hot moist sample—cold dry countercurrent shell flow, and    -   Cold moist sample—warm dry countercurrent shell flow.

The moisture exchange systems that can be used are manufactured by thePerma Pure, LLC corporation. An example is Model PH150-11060T-301 with60 tubes and is about 40 cm long and 5 cm in diameter for conditioningbioaerosol flows of 25 liters per minute. The purge or jacket flow ismaintained at 50 liters per minute.

In general, the controlled bioaerosol collection of this inventionpermits control of relative humidity during heating or cooling topreserve microorganisms by way of temperature gradients and/or RHgradients along a membrane-based moisture exchange system to controlrelative humidity during heating or cooling. The controlled bioaerosolcollection of this invention permits the use of multiple cascadedmembrane-based moisture exchange systems to control relative humidityduring heating or cooling with different schemes for particular flowsabout the membrane elements.

In one embodiment of this invention, a membrane-based moisture exchangesystem creates a controlled relative humidity for bioaerosol sampling.Use of this moisture exchange system prevents cross contaminationbecause the water is exchanged by a chemical process.

The control bioaerosol collection of this invention permits use of asample by-pass in a bioaerosol sampling system. The sample by-pass isrequired to allow the system to reach desired temperature and relativehumidity. When the appropriate bioaerosol conditions are reached at thebioaerosol collector, the sample flow can be switched from a by-passmode to a sample collection mode to initiate obtaining a bioparticlesample.

Once the samples are collected, the temperature and RH humidityconditions are controlled as noted above to maintain the viability ofthe collected bioaresol organism. Further, the collection media can berelocated from the bioaerosol sampling system to a temperature andhumidity controlled storage unit for transport or storage.

Experimental Evaluation

Three bioaerosol sampler prototypes following different embodiments ofthis invention have been used to evaluate particle collection,temperature and humidity conditioning, and maintenance of viabilityduring sampling. For these tests, the viability is measured bycomparison to a conventional all glass impinger (AGI) noted above. Thethree prototype samplers tested were:

-   -   (1) A “1-stage RH-conditioned biosampler” shown schematically in        FIG. 24A, and implemented as shown in FIG. 25A. This system        samples air at 25 L/min and is operated at ambient temperature        and controls filter RH to approximately 80% at ambient        temperature.    -   (2) A “refrigerated 1-stage RH-conditioned biosampler” which is        the second stage of the biosampler shown schematically in FIG.        24B, and implemented as shown in FIG. 25B. This system samples        air at 25 L/min and cools the sampled air stream to        approximately 11° C. and achieves at filter RH of approximately        75% using a Nafion unit and an air stream chilled by ice water.    -   (3) A “2-stage RH-conditioned biosampler” shown schematically in        FIG. 24B, and implemented as shown in FIG. 25C. This is a 25        L/min system that uses a two-stage system of Nafion units to        control the RH and temperature at the filter. Automated control        of the first stage provides consistent RH at the inlet of the        second stage that then cools the sample temperature to        approximately 10° C. and provides an RH of approximately 80% at        the filter.

Physical evaluation of the systems included measurement of particlepenetration through the Nafion conditioner, and of the robustness of therelative humidity and temperature control. Biological or viabilitytesting was done with four types of bioaerosols. These were Bacillusthuringiensis var. kurstaki (Btk), E. coli, MS2 phage, and Yersinia,where Btk is the hardiest, and Yersinia the most fragile. A bioaerosolchallenge of 10 minutes or 30 minutes was used followed by either noadditional exposure or by sampling of clean air for several hours. Asnoted above, an all glass impinge (AGI) is used during the bioaerosolchallenge time as a reference sample.

1-Stage Conditioning and Refrigerated 1-Stage RH-Conditioned Biosamplers

RH and Temperature Conditioning:

To test the performance of the conditioning system, the relativehumidity at the point of the filter collector was measured over widelyvarying values of the relative humidity of the sampled air stream. FIG.25D shows the results of these tests for the 1-stage conditioningbiosampler. As the input relative humidity was decreased from around 90%to below 5%, the relative humidity measured at the sample filterremained almost constant close to 78%. This relative humidity controlwas achieved through an automated feedback system that adjusted thevalve labeled “automated valve” in FIG. 25A to change the absolutepressure of the nearly-saturated flow in the shell space of the Nafionconditioner based on the value of the relative humidity at the samplefilter as measured by the sensor labeled “RH sensor” in FIG. 25A.

Viability for Sampling:

Ability to maintain the viability of airborne organism during collectionwas evaluated using E. coli, wherein an AGI sampler served as reference.Consecutive, 30-min samples were collected with a pair of biosamplersand with the AGI operating in parallel. AGI samples were removed fromthe sampling line immediately, while one of the pair of samples fromeach biosampler was then subjected to another 16 to 24 hours of cleanair sampling prior to being removed from the sampler. Comparison ofviable collection for the runs with, and without multi-hour clean airexposure shows the viability of the E. coli during collection. Table 5shows these results for several runs for the 1-stage RH-conditionedbiosampler and the refrigerated 1-stage RH-conditioned biosampler, bothreferenced to the AGI. For a 30-minute challenge, significant recoveryof E. coli is obtained for both biosamplers. For the 16-26 hr exposuresfollowing collection, the refrigerated sampler provided better recovery.

TABLE 5 E. Coli data comparing 1-stage RH-conditioned biosampler to theRefrigerated 1-Stage RH-conditioned BioSampler Refrigerated 1-Stage RH-1-Stage RH- conditioned conditioned biosampler biosampler Sampling % of% of Test time CFU/L air AGI CFU/L air AGI Run 1 30 min 1.0 × 10³ 1031.0 × 10³ 118 16.3 hr  2.0 × 10⁻² 0  2.0 × 10⁻² 0.09 Run 2 30 min 1.2 ×10³ 76 1.2 × 10³ 54 30 min 1.5 × 10³ 77 1.5 × 10³ 91 16 hr  2.7 × 10⁻²0.001  2.7 × 10⁻² 0.99 Run 3 30 min 1.2 × 10³ 92 1.2 × 10³ 61 30 min 2.3× 10³ 130 2.3 × 10³ 168 16 hr  3.3 × 10⁻² 0.001  3.3 × 10⁻² 3.8 Run 4 30min 7.8 × 10² 56 24 hr  3.5 × 10⁻² 0.02 Run 5 30 min 8.9 × 10² 38 24 hr 8.0 × 10⁻² 0.003 Run 6 30 min 2.0 × 10³ 91 24 hr  4.7 × 10⁻² 0.0022-Stage Conditioning Biosampler

RH and Temperature Conditioning:

To test the performance of the 2-Stage conditioning biosampler, thetemperature and relative humidity at the point of the filter collectorwas measured over widely varying values of the relative humidity of thesampled air stream. FIG. 25E show the temperature and humidity dataattained in these laboratory tests. The first Nafion conditioning stageoperated at ambient temperature. The shell pressure was controlled tobring the humidity of the exiting aerosol—the “Midpoint RH”—to a targetof 45% RH. This conditioned flow entered the second Nafion conditioningstage, which had a cold shell flow at fixed humidity. The exiting samplehumidity from this second stage is labeled “Sample RH.” In FIG. 25E, theinput relative humidity was rapidly changed from somewhat less than 10%to around 55%, and yet the relative humidity at the collection filterremained steady at around 82%.

Aerosol Penetration Tests:

Aerosol penetration through the 2-stage conditioning biosampler wastested using 2-μm fluorescent labeled polystyrene latex (PSL), withcomparison to a sampling tube with 37-mm PTFE air sampling filter. Theflow rates of 2-Stage Conditioning BioSampler and the 37-mm referencesampling filter were both 25 L/min. Table 6 reports the results of threeexperiments with PSL aerosol. Overall, the recovery of PSL from theNanofiber filters sampled with the 2-stage conditioning biosampler waslower than the recovery from Teflon filters sampled with the bare tube.

TABLE 6 Recovery of florescent 2 μm PSL aerosol for Nanofiber filersused in 2-stage conditioning biosampler compared to standard Teflon airsampling filter (reference filter). 2-Stage Teflon ConditioningReference Biosampler- Filter Nanofiber NF % of Experiment (FSU) Filter(FSU) Ref. Run #1 4.8 4.0 82% Run #2 10.5 4.5 43% Run #3 11.4 9.7 85%

Bioaerosol Contamination Testing:

Carryover or cross-contamination from one sample to the next was alsoinvestigated. Due to its hardiness, Bacillus thuringiensis var. kurstaki(Btk) was selected for this testing. Btk represents a design-limitingcase where a hardy organism collects within the sampler and then couldbecome re-entrained and deposited onto the collection filter during alater sampling time, thus contaminating the sampling filter. The testwas conducted by challenging with Btk for 30 minutes followed bydisconnecting 2-stage conditioning biosampler from the test rig,clearing the sampling line (about 1 minute of sampling the room air). Aclean filter was installed, and sampling commenced for 30 minutes tocollect potential re-entrained organisms. Table 7 summarizes the resultsfor this experiment.

TABLE 7 Measurement of re-entrainment of Btk using 30 min challenge,filter change out, and 30 min of clean air. A minimal amount of organismwas recovered indicating only minor carryover. CFU/L air ReferenceExperiment recovered to AGI 30 min bioaerosol challenge 94.3 117.6%Clean filter + 30 clean air 0.11 N.A.

Effect of Filter Quartering:

The effect of quartering filters (cutting into four quadrants) wasstudied with E. coli. A 10-minute challenge of aerosol was used with noexposure to clean air for the cases of whole filters versus quarteredfilters. The AGI was kept as a baseline for recovery of viable microbes.Table 8 summarizes the results. Table 8 presents the quartered filterdata at recovery from whole filter processing, summation of all quartersof a single filter, and processing of each quarter. Interestingly,quartering the filter significantly deteriorates the viable recovery.

TABLE 8 Effect of quartering of filters using E coli as the testorganism. Filters are challenged for 10 minutes with bioaerosol and thenprocessed (no clean air exposure). Whole filters and quarter filters arecompared by total organisms recovered relative to the AGI. Quarteredfilter data is presented as by individual piece and sum of the parts.Quartering filters significantly deteriorates recovery of microbes.Reference Experiment CFU/L air to AGI Whole-#1 87  95% Whole-#2 164112%  Quartered-#3-Sum 18  8% #3-Q1 9.0 4.0% #3-Q2 7.7 3.4% #3-Q3 1.00.45%  #3-Q4 0 0.0% Quartered-#4-Sum 56  19% #4-Q1 22 7.1% #4-Q2 10 3.5%#4-Q3 0.67 0.23%  #4-Q4 23 8.1%

Viability for Sampling Btk.

Btk results for the 2-stage conditioning BioSample are shown in Table 9.Recovery of viable Btk is accomplished in reasonable quantities.However, some loss relative to the AGI is observed. Previous experimentshad shown that extraction of bacillus spores can be inefficient, likelydue to interactions between the spore coat and the polymer nanofibers ofthe sampling filter. The longer aerosol challenge, 30 minutes, gave morefavorable results. The longer challenge time provides more organismsloaded onto the sampler filter but does not change the relative amountsof bioaerosol collected by the 2-stage conditioning biosampler relativeto the AGI.

TABLE 9 Results of Btk bioaerosol sampling by the 2-Stage ConditioningBioSampler prototype compared to the AGI reference. Recovery of Btk isachieved even with 4 hrs of clean air exposure but is, at most,approximately half that initially recovered by the AGI. Organism Cleanair Refer- Filter Sample Time Sample Time CFU/L ence process- Experiment(minutes) (hours) air to AGI ing Jul. 12, 2012 30 4 67 56% whole Jul.23, 2012 10 4 5.3 26% whole Jul. 25, 2012 10 4 5.6 22% whole

Viability for Sampling E. coli.

Results for recovery of E. coli are summarized in Table 10. Severaldifferent experiments were conducted looking at the effect of challengetime, clean air exposure time, and filter quartering. Most results aresingle data points unless noted as an average. For a 30-minutechallenge, significant recovery of E. coli is obtained indicatingsimilar results to Btk for comparing 30-min and 10-min challenge times.A single 16-hr clean air challenge was conducted and the results, 0.5%viable recovery, compared well with previous results from therefrigerated RH-conditioned biosampler under the same conditions: 0.1%to 4% of the AGI for the Refrigerated RH-conditioned BioSampler. A 4-hrclean air challenge resulted in minimal loss of viability (compare 0 and4-hr clean air challenges in Table 9).

TABLE 10 Results of E. coli bioaerosol sampling for various experimentalconditions. Viable recovery is best for a whole filter and 4 hrs ofclean air challenge. Organism Clean air Refer- Sample Time Sample TimeCFU/L ence Filter Experiment (minutes) (hours) air to AGI type 30 min 300 1567 153% Whole whole 30 min 30 16 5.4 0.49%  Whole whole + air 10 min10 0 126 103% Whole whole (average two samples) 10 min 10 4 286 146%Whole whole + air

Control of the Bioaerosol Collection System

Control of the bioaerosol collection system of this invention istypically under processor control. FIG. 32 illustrates a computer system1201 for implementing various embodiments of this invention. Thecomputer system 1201 may be used as the computer system 5 to perform anyor all of the control functions described above to regulate gas flow,temperature and humidity. The computer system 1201 includes a bus 1202or other communication mechanism for communicating information, and aprocessor 1203 coupled with the bus 1202 for processing the information.The computer system 1201 also includes a main memory 1204, such as arandom access memory (RAM) or other dynamic storage device (e.g.,dynamic RAM (DRAM), static RAM (SRAM), and synchronous DRAM (SDRAM)),coupled to the bus 1202 for storing information and instructions to beexecuted by processor 1203. In addition, the main memory 1204 may beused for storing temporary variables or other intermediate informationduring the execution of instructions by the processor 1203. The computersystem 1201 further includes a read only memory (ROM) 1205 or otherstatic storage device (e.g., programmable ROM (PROM), erasable PROM(EPROM), and electrically erasable PROM (EEPROM)) coupled to the bus1202 for storing static information and instructions for the processor1203.

The computer system 1201 also includes a disk controller 1206 coupled tothe bus 1202 to control one or more storage devices for storinginformation and instructions, such as a magnetic hard disk 1207, and aremovable media drive 1208 (e.g., floppy disk drive, read-only compactdisc drive, read/write compact disc drive, compact disc jukebox, tapedrive, and removable magneto-optical drive). The storage devices may beadded to the computer system 1201 using an appropriate device interface(e.g., small computer system interface (SCSI), integrated deviceelectronics (IDE), enhanced-IDE (E-IDE), direct memory access (DMA), orultra-DMA).

The computer system 1201 may also include special purpose logic devices(e.g., application specific integrated circuits (ASICs)) or configurablelogic devices (e.g., simple programmable logic devices (SPLDs), complexprogrammable logic devices (CPLDs), and field programmable gate arrays(FPGAs)).

The computer system 1201 may also include a display controller 1209coupled to the bus 1202 to control a display 1210, such as a cathode raytube (CRT), for displaying information to a computer user. The computersystem includes input devices, such as a keyboard 1211 and a pointingdevice 1212, for interacting with a computer user and providinginformation to the processor 1203. The pointing device 1212, forexample, may be a mouse, a trackball, or a pointing stick forcommunicating direction information and command selections to theprocessor 1203 and for controlling cursor movement on the display 1210.

The computer system 1201 performs a portion or all of the controlfunctions described above by having instructions programmed to performvarious steps for control of the humidifier, air stream, refrigeration,RH, and temperature gradients. Such instructions may be read into themain memory 1204 from another computer readable medium, such as a harddisk 1207 or a removable media drive 1208. One or more processors in amulti-processing arrangement may also be employed to execute thesequences of instructions contained in main memory 1204. In alternativeembodiments, hard-wired circuitry may be used in place of or incombination with software instructions. Thus, embodiments are notlimited to any specific combination of hardware circuitry and software.

As stated above, the computer system 1201 includes at least one computerreadable medium or memory for holding instructions programmed accordingto the teachings of the invention and for containing data structures,tables, records, or other data described herein. Examples of computerreadable media are compact discs, hard disks, floppy disks, tape,magneto-optical disks, PROMs (EPROM, EEPROM, flash EPROM), DRAM, SRAM,SDRAM, or any other magnetic medium, compact discs (e.g., CD-ROM), orany other optical medium, punch cards, paper tape, or other physicalmedium with patterns of holes, a carrier wave (described below), or anyother medium from which a computer can read.

Generalized Aspects of the Invention

In one aspect of the invention, an aerosol collection system includes abio-aerosol delivery device configured to supply bioparticles in a gasstream, a moisture exchange device including a partition member coupledto the gas stream and configured to humidify or dehumidify thebioparticles in the gas stream with a vapor of a biocompatible liquid(e.g., water or other non-toxic fluid), and an aerosol collection mediumdownstream from the moisture exchange device and configured to collectthe bioparticles.

In this aspect, the moisture exchange device can be at least one of apermeable material, a semi-permeable membrane material, or a polymericionomer. The moisture exchange device include least one of:

-   -   a copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic        acid and tetrafluoroethylene;    -   polyethylene oxide, polyvinyl alcohol, cellulose ether and        starch) and copolymers thereof; and    -   polyacrylic acid, polyacrylamide, polyisopropyl acrylamide,        polystyrene sulfonic acid, polyvinyl pyridine and polyamino acid        and copolymers and salts thereof.

The moisture exchange device include tubes of at least one of apermeable material, a semi-permeable membrane material, or a polymericionomer. The moisture exchange device include tubes of at least one of:

-   -   a copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic        acid and tetrafluoroethylene;    -   polyethylene oxide, polyvinyl alcohol, cellulose ether and        starch) and copolymers thereof; and    -   polyacrylic acid, polyacrylamide, polyisopropyl acrylamide,        polystyrene sulfonic acid, polyvinyl pyridine and polyamino acid        and copolymers and salts thereof.

In this aspect, the aerosol collection system can include a by-pass gasflow device configured to supply air flow without the entrainedbioparticles in order stabilize at least one of a temperature andrelative humidity of the aerosol collection medium prior to collectingthe bioparticles.

In this aspect, the aerosol collection system can include a controllerconfigured to control temperature and a relative humidity along agas-flow axis of the membrane-based moisture exchange device. Thecontroller can be programmed to adjust the temperature and the relativehumidity such that the particles in the gas stream transition fromoutside ambient conditions to a target temperature and relative humiditycondition. The target temperature and relative humidity condition can becontrolled to be in at least one of the following conditions:

4° C., and RH of 5 to 95%;

2° C. to 6° C., and RH of 5 to 95%;

1° C. to 8° C., and RH of 5 to 95%; or

2° C. to 10° C., and RH of 5 to 95%.

The controller can be programmed to adjust a temperature and a relativehumidity of the bioparticle while transitioning from outside ambientconditions to the target temperature and relative humidity conditionalong a controlled path of temperature and humidity. The controlled pathmaintains a relative humidity level greater than 5%, greater than 10%,greater than 15%, greater than 25%, or greater than 50%, duringtransport of the bioparticles to the collection medium.

With the conditioned biosamplers of this invention, the targettemperature and relative humidity condition can be controlled to be inat least one of the following conditions:

1° C. to 10° C., and RH of 50 to 95%;

1° C. to 10° C., and RH of 60 to 90%; or preferably

1° C. to 10° C., and RH of 70 to 85%; or more preferably

2° C. to 12° C., and RH of 70 to 85%.

Alternatively, if sampling in an indoor environment, with retrieval ofthe sample shortly after collection, then it is possible to control thehumidity condition of the sampling. In this instance, active temperaturecontrol may not be needed, and instead the humidity only is controlled.The relative humidity condition can be controlled to be in one of thefollowing conditions:

RH of 50% to 95%;

RH of 60% to 90%; or preferable

RH of 70% to 85%; or more preferably

RH of 75% to 85%.

In this aspect, the aerosol collection medium can be at least one of aflow-through or an impaction device including a plurality of fibers.

In this aspect, the aerosol collection medium can include a viabilityenhancing material provider disposed in a vicinity of the plurality offibers and configured to provide a viability enhancing material to thecollected bioparticles to maintain viability of the collectedbioparticles. The viability enhancing material provider can include anosmotic material disposed in contact with the plurality of fibers andconfigured to maintain a relative humidity suitable for said viabilityof bioparticles. The osmotic material can be a water-regulating materialconfigured to provide water and/or a nutrient supply to the fibers tosupport biological viability of the collected bioparticles. The nutrientsupply can include a supply of at least one of water, proteins,carbohydrates, sugars, salts, phosphate buffered saline, and tryptic soybroth.

In this aspect, the aerosol collection medium can be a plurality offibers formed into a fiber mat for collection of the bioaerosolparticles. The plurality of fibers can have an average fiber diameter ofless than 10 microns. The plurality of fibers can have an average fiberdiameter of less than 1 micron. The plurality of fibers can have anaverage fiber diameter of less than 0.5 micron.

In this aspect, the biocompatible liquid can include solutions includingat least one or more of water, proteins, carbohydrates, sugars, salts,phosphate buffered saline, and tryptic soy broth.

In another aspect of the invention, a method for collecting aerosolsincludes delivering bioparticles in an gas stream, humidifying ordehumidifying the bioparticles in the gas stream by transport of wateror other non-toxic fluid across a partition member and into a vaporphase of the gas stream, and collecting the bioparticles by a collectionmedium.

The humidifying or dehumidifying can maintain a relative humiditygreater than 5%, greater than 10%, greater than 15%, greater than 25%,or greater than 50%. The humidifying or dehumidifying can transport thebiocompatible liquid across at least one of a permeable material, asemi-permeable membrane material, or a polymeric ionomer. Thehumidifying or dehumidifying can transport the biocompatible liquidacross at least one of:

-   -   a copolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic        acid and tetrafluoroethylene;    -   polyethylene oxide, polyvinyl alcohol, cellulose ether and        starch) and copolymers thereof; and    -   polyacrylic acid, polyacrylamide, polyisopropyl acrylamide,        polystyrene sulfonic acid, polyvinyl pyridine and polyamino acid        and copolymers and salts thereof.

In this aspect, the method for collecting aerosols can stabilize thetemperature and/or the relative humidity of the aerosol collectionmedium prior to collecting the bioparticles. In this aspect, the methodfor collecting aerosols can control a temperature and a relativehumidity along a gas-flow axis of the membrane.

In this aspect, the method for collecting aerosols can adjust thetemperature and the relative humidity such that the particles in the gasstream transitions from outside ambient conditions to a targettemperature and relative humidity condition. In this aspect, the methodfor collecting aerosols can adjust the temperature and the relativehumidity to at least one of the following conditions:

4° C., and RH of 5 to 95%;

2° C. to 6° C., and RH of 5 to 95%;

1° C. to 8° C., and RH of 5 to 95%; or

2° C. to 10° C., and RH of 5 to 95%.

Other conditions for collecting aerosols include the adjusting of therelative humidity at the point of sampling to range between 50% and 90%and more preferably between 75% to 85% range both at ambient collectiontemperature (e.g., ˜20-25° C.) and at cooled temperatures (e.g., 4-10°C.).

Accordingly, in one embodiment, the present invention provides forviable collection of bioaerosol through control of the relative humidityat the point of sampling to the range of 75% to 85%. Collection at thistarget relative humidity range can provide viable sampling at ambienttemperatures, or at refrigerator temperatures, where refrigeratortemperatures are defined as in the range between 1° C. and 10° C. Whenthere is a need to store the sample for an extended time, sampling canoccur at refrigerator temperatures.

The embedded results below show the percentage recovery of viralsimulants as compared to collection of these simulants under “normal”room temperature and humidity conditions were these simulants normallydoe not survive and are not recoverable. Collecting at the higherhumidity conditions improves the recoverability.

Conditions % Recovered RH T (C.) Yersinia r. Serratia 40% 22 0%  0% 80%22 0% 11% 80% 10 0.2% to 20% not tested

Organism % Recovered RH T Bacillus E coli Serratia Yersinia MS2 80% 22°C. 30 ± 17% N.T. 11% 0% N.T. 80% 10° C. 79 ± 5%  40 ± 20% N.T. 0.2 to20% 11 ± 7% N.T. = not tested

In this aspect, the method for collecting aerosols can adjust atemperature and a relative humidity of the bioparticle whiletransitioning from outside ambient conditions to the target temperatureand relative humidity condition along a controlled path of temperatureand humidity, where the controlled path maintains a relative humiditylevel greater than 5%, greater than 10%, greater than 15%, greater than25%, or greater than 50%, during transport of the bioparticles to thecollection medium.

In this aspect, the method for collecting aerosols can provide aviability enhancing material to the collection medium such as forexample water, proteins, carbohydrates, sugars, salts, phosphatebuffered saline, and/or tryptic soy broth.

In this aspect, the method for collecting aerosols can collect at leastone of pox viruses, filoviruses, arenaviruses, alphavirus, brucellaspecies, burkholderia mallei, yersinia pestis and coxiella burnetii onthe collection medium.

In another aspect of the invention, a collection device includes aplurality of fibers formed into a fiber mat. The fiber mat is configuredto collect and maintain the viability of microbes and/or bioparticles.The fibrous filter can be configured in any manner used in airsampling/aerosol collection using a flow-through filter. In one aspectof the invention, the fibers are configured as an impaction substrate tocollect and maintain microbes and/or bioparticles for use in airsampling/aerosol collection using the method of impaction. The fibrousfilter can be configured in any manner used for swabbing or the wipingof surfaces or the sampling of bioparticles in liquids.

In another aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and configured to collect bioparticlesthereon, and including a viability enhancing material provider disposedin a vicinity of the plurality of fibers and configured to provide aviability enhancing material to the collected bioparticles to maintainviability of the bioparticles collected by the fiber mat. In this aspectof the invention, the viability enhancing material may or may not be asubset of the plurality of fibers.

In one aspect of the invention, the filter or impaction substrateincludes a fibrous mat configured in terms of structure, surfacechemistry, and additives to provide enhanced support of viabilitymaintenance of the bioparticles collected.

In one aspect of the invention, a filtration or impaction device foraerosol collection includes a fibrous mat and in conjunction with amechanism or method for conditioning the moisture content of the airentering the air sampling device to a value that provides enhancedcollection and maintenance of the bioparticles collected.

In one aspect of the invention, the air flow containing bioaerosol isconditioned as to relative humidity and temperature, and the particlestherein are collected by impaction onto a fibrous substrate or by asubsequent filtration mechanism. The fibrous substrate and/or thesubsequent filtration mechanism provide a collection mechanism ofbioparticles and provide a mechanism for maintenance of viability of thecollected bioparticles.

In one aspect of the invention, the aerosol is exposed to the vapor or aworking fluid (such as for example water and other fluids that arebiocompatible, possibly including silicone fluids) in a saturationchamber. Subsequently, vapor condensation onto particles is induced byeither adiabatic expansion or cooling in the condensing chamber, or bymixing with a cooler airflow. The enlarged particles are subsequentlycollected via impaction or filtration on a nanofiber or fiber material.

In one aspect of the invention, a fibrous mat is configured to provideenhanced recovery of the collected material. Enhanced recoveryincludes 1) recovery of the particles such that the extraction proceduredoes not decrease their viability; 2) a collection and extraction whichdoes not prevent or impede subsequent analysis such as live culture,PCR-based techniques, or any other chemical or physical analysis of thecollected material/organisms; and 3) enhanced release of the collectedmaterial through dissolution of the fibrous material using selectsolvents and/or processing conditions.

In one aspect of the invention, the fibers in the fibrous material orthe fiber mat have an average fiber diameter of less than 10 μm, or lessthan 1 μm, or less than 500 nm, or less than 300 nm, or less than 200nm, or less than 100 nm.

In another aspect of the invention, there is provided a method forcollecting aerosols. This method includes entraining particles in a gasstream, exposing the particles in the gas stream to a solvent, andcollecting the aerosol particles by a collection medium. The collectionmedium includes a plurality of fibers formed into a fiber mat includingand a viability enhancing material provider disposed in a vicinity ofthe plurality of fibers and configured to provide a viability enhancingmaterial to the collected bioparticles to maintain viability of thebioparticles collected by the fiber mat.

This method also can inject the viability enhancing material into thecollection medium prior to collecting the aerosol particles. Theviability enhancing material injected can be at least one of water,proteins, carbohydrates, sugars, salts, phosphate buffered saline, andtryptic soy broth. This method also can inject the viability enhancingmaterial (such as those listed above) into the collection medium duringthe collecting of the aerosol particles. This method also can injectantioxidants such as for example nitrous oxide (NO) into the collectionmedium.

This method also can introduce an agent to reduce oxygen toxicity to thebioparticles collected in the collection medium. Such an agent caninclude enzymes or fullerenes to reduce oxygen toxicity.

In another aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and an osmotic material disposed incontact with the plurality of fibers. The osmotic material can be aviability enhancing material configured to maintain viability ofbioparticles collected by the fiber mat. The osmotic material can be awater-regulating material configured to provide water to the fibers. Theosmotic material can constitute a nutrient supply providing nutrients tosupport biological viability of biomaterial collected in the filtrationmedium. The nutrient supply can be at least one of water, proteins,sugars, carbohydrates, salts, phosphate buffered saline, and tryptic soybroth.

The collection medium and the viability enhancing material can bedisposed in one of an air filter, a wipe, a brush, a swab, a sorbentpad, or a liquid filter. The fibers can be made of materials which aredissolvable in a bio-compatible solvent.

The collection medium can include a support (e.g., a rigid support)supporting the collection medium. The support can be one of a filter, aplastic foam, a metallic foam, a semi-conductive foam, a woven material,a nonwoven material, a plastic screen, a textile, and a high efficiencyparticulate air (HEPA) filter medium.

In another aspect of the invention, there is provided an aerosolcollection system including an aerosol pumping device configured toentrain particles in a gas stream, an aerosol saturation deviceconfigured to expose the particles in the gas stream with abiocompatible liquid, and an aerosol collection medium downstream fromthe aerosol saturation device. The aerosol collection medium includes aplurality of fibers formed into a fiber mat for collection of theaerosol particles, and an osmotic material disposed in contact with theplurality of fibers.

The aerosol collection system can include a humidity control deviceconfigured to maintain the collection medium at a relative humidity from50 to 100%, or at a relative humidity from 65 to 85%, or at a relativehumidity from 75 to 81%.

The aerosol collection medium in this aspect of the invention can be atleast one of a flow-through or an impaction device. The osmotic materialin this aspect of the invention can be a viability enhancing materialconfigured to maintain viability of bioparticles collected by the fibermat. The osmotic material in this aspect of the invention can be awater-regulating material configured to provide water to the fibers. Theosmotic material in this aspect of the invention can be a nutrientsupply providing nutrients to support biological viability ofbiomaterial collected in the filtration medium. The nutrient supply inthis aspect of the invention can be a supply of at least one ofproteins, sugars, and salts.

The fibers in this aspect of the invention can be nanofibers, can beformed of materials dissolvable in a bio-compatible solvent. A support(rigid or not) can be used to support the collection medium. The supportin this aspect of the invention can be at least one of a filter, aplastic foam, a metallic foam, a semi-conductive foam, a woven material,a nonwoven material, a plastic screen, a textile, and a high efficiencyparticulate air (HEPA) filter medium.

In another aspect of the invention, there is provided a method forcollecting aerosols. The method included entraining particles in an gasstream, exposing the particles in the gas stream to a biocompatibleliquid, and collecting the aerosol particles by a collection mediumincluding a plurality of fibers formed into a fiber mat including and anosmotic material disposed in contact with the plurality of fibers.

This method also can inject the viability enhancing material into thecollection medium prior to collecting the aerosol particles. Theviability enhancing material injected can be at least one of water,proteins, carbohydrates, sugars, salts, phosphate buffered saline, andtryptic soy broth. This method also can inject the viability enhancingmaterial (such as those listed above) into the collection medium duringthe collecting of the aerosol particles. This method also can injectantioxidants such as for example nitrous oxide (NO) into the collectionmedium.

This method also can introduce an agent to reduce oxygen toxicity to thebioparticles collected in the collection medium. Such an agent caninclude enzymes or fullerenes to reduce oxygen toxicity.

In another aspect of the invention, there is provided a bioparticlecollection device including a collection medium including a plurality offibers formed into a fiber mat and an osmotic material disposed incontact with the plurality of fibers. The osmotic material can be aviability enhancing material configured to maintain viability ofbioparticles collected by the fiber mat. The osmotic material can be awater-regulating material configured to provide water to the fibers. Theosmotic material can constitute a nutrient supply providing nutrients tosupport biological viability of biomaterial collected in the filtrationmedium. The nutrient supply can be at least one of water, proteins,sugars, carbohydrates, salts, phosphate buffered saline, and tryptic soybroth.

The collection medium and the viability enhancing material can bedisposed in one of an air filter, a wipe, a brush, a swab, a sorbentpad, or a liquid filter. The fibers can be made of materials which aredissolvable in a bio-compatible solvent.

The bioparticle collection device can include a support (e.g., a rigidsupport) supporting the collection medium. The support can be one of afilter, a plastic foam, a metallic foam, a semi-conductive foam, a wovenmaterial, a nonwoven material, a plastic screen, a textile, and a highefficiency particulate air (HEPA) filter medium.

Numerous modifications and variations of the invention are possible inlight of the above teachings. It is therefore to be understood thatwithin the scope of the appended claims, the invention may be practicedotherwise than as specifically described herein.

The invention claimed is:
 1. A method for collecting aerosols,comprising: providing an aerosol humidifying device having therein a) afirst temperature and humidity controllable gaseous environment and b) asecond temperature and humidity controllable gaseous environment, thefirst and second temperature and humidity controllable gaseousenvironments separated only by a permeable partition member having apolymeric wall; delivering, as the aerosols to be collected,bioparticles from outside into a first gas stream of the firsttemperature and humidity controllable gaseous environment; humidifyingor dehumidifying the bioparticles in the first gas stream of the firsttemperature and humidity controllable gaseous environment by transportof water from a second gas stream of the second temperature and humiditycontrollable gaseous environment across the permeable partition memberand into a vapor phase of the first gas stream, the permeable partitionmember preventing contamination between a) the first gas stream of thefirst temperature and humidity controllable gaseous environmentincluding the bioparticles and b) the second gas stream of the secondtemperature and humidity controllable gaseous environment; andcollecting the bioparticles by a collection medium.
 2. The method ofclaim 1, wherein humidifying or dehumidifying the particles comprisesmaintaining a relative humidity greater than 5%.
 3. The method of claim1, wherein said polymeric wall comprising at least one of a permeablematerial, a semi-permeable membrane material, or a polymeric ionomer,and the polymeric wall surrounding surrounds the first gas streamincluding the bioparticles.
 4. The method of claim 1, whereinhumidifying or dehumidifying the particles comprises transporting thewater through the said polymeric wall comprising at least one of: acopolymer of perfluoro-3,6-dioxa-4-methyl-7octene-sulfonic acid andtetrafluoroethylene; polyethylene oxide, polyvinyl alcohol, celluloseether and starch) and copolymers thereof; and polyacrylic acid,polyacrylamide, polyisopropyl acrylamide, polystyrene sulfonic acid,polyvinyl pyridine and polyamino acid and copolymers and salts thereof.5. The method of claim 1, further comprising: stabilizing at least oneof a temperature and relative humidity of the aerosol collection mediumprior to collecting the bioparticles.
 6. The method of claim 1, furthercomprising: controlling a temperature and a relative humidity along agas-flow axis of the membrane.
 7. The method of claim 6, furthercomprising: adjusting the temperature and the relative humidity suchthat the particles in the first gas stream transition from outsideambient conditions to a target temperature and relative humiditycondition.
 8. The method of claim 7, further comprising: adjusting thetemperature and the relative humidity to one of the following sets ofconditions: 2° C. to 10° C. and RH of 5 to 95% 2° C. to 10° C. and RH of70 to 85%; 1° C. to 8° C. and RH of 5 to 95%; 1° C. to 8° C. and RH of70 to 85%; 2° C. to 6° C. and RH of 5 to 95%; 2° C. to 6° C. and RH of70 to 85%; 4° C. and RH of 5 to 95%; or 4° C. and RH of 70 to 85%. 9.The method of claim 7, further comprising: adjusting a temperature and arelative humidity of the bioparticle while transitioning from outsideambient conditions to the target temperature and relative humiditycondition along a controlled path of temperature and humidity, saidcontrolled path comprises a two-stage system including a first stagehaving a relative humidity in a range from 40-60% at ambient temperatureand a second stage below ambient temperature while maintaining therelative humidity in said range from 40-60%.
 10. The method of claim 1,further comprising: providing a viability enhancing material to thecollection medium.
 11. The method of claim 1, wherein providing aviability enhancing material comprises providing at least one of water,proteins, carbohydrates, sugars, salts, phosphate buffered saline, andtryptic soy broth to the collection medium.
 12. The method of claim 1,wherein collecting the bioparticles comprises collecting at least one ofpox viruses, filoviruses, arenaviruses, alphavirus, brucella species,burkholderia mallei, yersinia pestis and coxiella burnetii on thecollection medium.
 13. The method of claim 1, wherein said polymericwall comprises at least one tubular wall separating the firsttemperature and humidity controllable gaseous environment from thesecond temperature and humidity controllable gaseous environment. 14.The method of claim 13, wherein said polymeric wall comprises pluraltubular walls separating the first temperature and humidity controllablegaseous environment from the second temperature and humiditycontrollable gaseous environment.
 15. The method of claim 1, whereinsaid polymeric wall comprises a flat sheet membrane separating the firsttemperature and humidity controllable gaseous environment from thesecond temperature and humidity controllable gaseous environment.